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Advances in Protein Chemistry and
Structural Biology Volume 115 - DNA

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Contributors
Salim Abdisalaam
Division of Molecular Radiation Biology, Department of Radiation Oncology, University of
Texas Southwestern Medical Center, Dallas, TX, United States
Aroumougame Asaithamby
Diana Azenha
Faculty of Pharmacy; Center for Neuroscience and Cell Biology and Institute for Biomedical
Imaging and Life Sciences (CNC.IBILI), University of Coimbra; Portuguese Institute for
Oncology at Coimbra, Coimbra, Portugal
Robert-Marlo Bautista
The Markey Cancer Center; Department of Surgery, University of Kentucky College of
Medicine, Lexington, KY, United States
Reena Beggs
Department of Radiation Oncology, University of Alabama-Birmingham School of
Medicine, Birmingham, AL, United States
Souparno Bhattacharya
Katharine M. Carter
The Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY,
United States
John A. D’Orazio
The Markey Cancer Center; Department of Toxiciology and Cancer Biology; Department
of Pediatrics, University of Kentucky College of Medicine, Lexington, KY, United States
C. George Priya Doss
Department of Integrative Biology, School of Bio Sciences and Technology, VIT, Vellore,
Tamil Nadu, India
Madeline Krentz Gober
United States
Anastas Gospodinov
Roumen Tsanev Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia,
Bulgaria
Sergi Guerrero Llobet
Department of Medical Oncology, University Medical Center Groningen, University of
Groningen, Groningen, The Netherlands
ix
x Contributors
Seref Gul
Department of Chemical and Biological Engineering, Koc University, Istanbul, Turkey
Nathaniel C. Holcomb
United States
Pablo Huertas
Departamento de Genetica, Universidad de Sevilla; Centro Andaluz de Biologı́a Molecular y
Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de
Olavide, Sevilla, Spain
Stuart G. Jarrett
The Markey Cancer Center; Department of Toxiciology and Cancer Biology, University of
Kentucky College of Medicine, Lexington, KY, United States
Sonia Jimeno
E. Judith
Tamil Nadu, India
Ibrahim Halil Kavakli
Department of Chemical and Biological Engineering; Department of Molecular Biology and
Genetics, Koc University, Istanbul, Turkey
Olga Kolesnikova
Institut de Genetique et de Biologie Moleculaire et Cellulaire Illkirch Cedex,
C.U. Strasbourg; Centre National de la Recherche Scientifique, UMR7104; Institut
National de la Sante et de la Recherche Medicale, U1258; Universite de Strasbourg, Illkirch,
France
Maria Celeste Lopes
Faculty of Pharmacy; Center for Neuroscience and Cell Biology and Institute for Biomedical
Imaging and Life Sciences (CNC.IBILI), University of Coimbra, Coimbra, Portugal
Teresa C. Martins
Center for Neuroscience and Cell Biology and Institute for Biomedical Imaging and Life
Sciences (CNC.IBILI), University of Coimbra; Portuguese Institute for Oncology at
Coimbra, Coimbra, Portugal
Fernando Mejı́as-Navarro
Enid Mendonca
Tamil Nadu, India
Contributors xi
Shibani Mukherjee
Nuri Ozturk
Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli,
Turkey
Arnaud Poterszman
Institut de Genetique et de Biologie Moleculaire et Cellulaire Illkirch Cedex, C.U.
Strasbourg; Centre National de la Recherche Scientifique, UMR7104; Institut National de la
Sante et de la Recherche Medicale, U1258; Universite de Strasbourg, Illkirch, France
Rosario Prados-Carvajal
J. Priyadharshini Christy
Tamil Nadu, India
Laura Radu
Institut de Genetique et de Biologie Moleculaire et Cellulaire Illkirch Cedex,
C.U. Strasbourg; Centre National de la Recherche Scientifique, UMR7104; Institut
National de la Sante et de la Recherche Medicale, U1258; Universite de Strasbourg, Illkirch,
France
Pepijn M. Schoonen
Debapriya Sinha
Kalayarasan Srinivasan
B. Susmita
Tamil Nadu, India
D. Thirumal Kumar
Tamil Nadu, India
Iva Ugrinova
Roumen Tsanev Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia,
Bulgaria
xii Contributors
Marcel A.T.M. van Vugt

Eddy S. Yang
Department of Radiation Oncology; Hugh Kaul Precision Medicine Institute, University of
Alabama-Birmingham School of Medicine, Birmingham, AL, United States
Hatem Zayed
Department of Biomedical Sciences, College of Health and Sciences, Qatar University,
Doha, Qatar
Preface
DNA is often under constant stress and damage. DNA damage is a biological
process that negatively impacts human health in many ways. Eukaryotic cells
accumulate DNA damage as a result of endogenous metabolic activities, such
as DNA replication and recombination errors, environmental exposures, such
as ionizing radiation, ultraviolet light, and chemical mutagens. There are dif-
ferent forms of damage and therefore diverse specialized repair mechanisms
take place to address the unique forms of damage. There are three types of
repair mechanisms: direct reversal of the damage, excision repair, and pos-
treplication repair. Direct reversal repair is specific to the damage. In contrast,
excision repair can be either specific or nonspecific. Postreplication repair
takes place downstream of the lesion due to replication being blocked at
the site of damage. DNA repair pathways and molecular machinery involved
in these pathways have been studied very extensively. Our comprehensive
knowledge in this field has been further employed in designing numerous
novel strategies for therapeutic treatment of different tumor types, some of
which proved to be superior compared to the conventional treatments.
This thematic volume focuses on the pathways for DNA repair and their
targeting as treatment strategies for cancer therapy. Exploitation of the DNA
repair machinery for personalized treatment and the mechanistic link
between DNA repair factors and immune signaling are also discussed.
Finally, examples of applying computational approaches for studying the
DNA repair mechanisms are given. This volume will be particularly useful
for researchers working on molecular mechanisms orchestrating DNA repair
and targeting these mechanisms as therapeutic strategies. This volume is
also intended as a scholarly material for use by students interested and/or
working on projects in the field.
DR. ROSSEN DONEV
MicroPharm Ltd
United Kingdom
xiii
CHAPTER ONE
DNA repair by photolyases

Ibrahim Halil Kavaklia,b,*, Nuri Ozturkc, Seref Gula
a
Department of Chemical and Biological Engineering, Koc University, Istanbul, Turkey
b
Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
c
Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey
*Corresponding author: e-mail address: hkavakli@ku.edu.tr
Contents
1. Introduction 1
2. Cryptochrome/photolyase protein family 2
2.1 Cofactors of the photolyases 5
2.2 The crystal structure of the photolyase 8
2.3 Reaction mechanism of DNA repair mediated by CPD photolyase 10
2.4 Reaction mechanisms of DNA repair mediated by (6-4) and other classes of
the photolyases 12
3. Conclusion 15
Acknowledgment 15
References 15
Abstract
Photolyases belong to the cryptochrome/photolyase protein family (CPF) which per-
form different functions such as DNA repair, circadian photoreceptor, and transcrip-
tional regulation. Photolyase is a flavoprotein that repairs UV-induced DNA damages
of cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6-4) photoproducts
using blue-light as an energy source. This enzyme has two chromophores: flavin ade-
nine dinucleotide (FAD) as a cofactor and a photoantenna such as methenyltetra-
hydrofolate (MTHF). The FAD is essential for catalysis of the DNA repair. The second
chromophore absorbs photons from the blue light spectrum and transfers energy to
FAD to increase the repair efficiency of the enzyme. Phylogenetic analysis in which
amino acid sequences of several hundreds of CPF members are used suggests that they
form more classes than we have considered so far. In this chapter, we discussed
structure-functions and reaction mechanisms of different classes of photolyases.
1. Introduction
DNA is the source of genetic information in all living cells. Therefore,
its stability and integrity are crucial to life. Considering its chemical nature it
can undergo several different modifications (induced by factors such as
#
Advances in Protein Chemistry and Structural Biology, Volume 115 2019 Elsevier Inc. 1
ISSN 1876-1623 All rights reserved.
https://doi.org/10.1016/bs.apcsb.2018.10.003
2 Ibrahim Halil Kavakli et al.
oxidative damage, ultraviolet and other types of radiation, and hydrolytic

damage), which can cause alterations in its composition. In addition, the
DNA sequence can also change during DNA replication despite proofread-
ing activity of the DNA polymerases. Modified/altered DNA structure
needs to be corrected in either case to prevent mutations and genomic insta-
bility of the cells. If these modifications and base changes permanently stay in
the genome of organisms they can lead to the development of cancers
depending on the nature of mutations. Therefore, cells possess different types
of the DNA repair mechanisms to minimize mutagenic changes. There
are mainly two types of DNA repair mechanisms, which are single strand
(ss)- and double strand (ds)-DNA damage repair. The ssDNA damage repair
mechanisms are further divided into direct reversal repair, nucleotide exci-
sion repair, base excision repair, and mismatch repair while dsDNA damage
repair mechanisms are divided into homologous recombination and non-
homologous end joining repairs. Among these, direct reversal repair mech-
anism does not require template information and corrects damages by simply
reversing them using DNA repair enzymes. Three major direct repair mech-
anisms are mediated by photolyases, O6-alkylguanine-DNA alkyltransferases
(AGTs), and the AlkB family dioxygenases, which reverse UV light-induced
photolesions, O-alkylated DNA damage, and N-alkylated base adducts,
respectively (Yi & He, 2013). In this chapter, we will highlight structure-
functions and reaction mechanisms mediated by photolyases.
2. Cryptochrome/photolyase protein family

The quality and quantity of ultraviolet (UV) radiation reaching the
earth’s surface from the sun depends on the transmission properties of the
atmosphere, which has changed over the course of time as a result of human
activity. For example, ozone layer thickness has been declining for a century,
which causes more UV to reach the earth’s surface (Bais et al., 2015).
UV irradiation induces DNA damage in the form of cyclobutane pyrimidine
dimers (Pyr<>Pyr, CPD, 80–90%) and pyrimidine-pyrimidone (6-4)
photoproducts (Pyr [6-4] Pyr, 10–20%) in organisms (Fig. 1) (Menck,
2002; Thompson & Sancar, 2002). This damage is reversed by DNA pho-
tolyases using blue-light (350–500 nm) as an energy source (Fig. 1) in some
organisms and by the nucleotide excision repair in others including those
with photolyases (Sancar, 2003). Molecular evolutionary analysis, genetic
and biochemical studies with photolyase-related genes, isolated from several
organisms throughout years, revealed that this family forms different classes
Photolyase 3
Fig. 1 Ultraviolet (UV) induces DNA damages and causes the formation of photoprod-
ucts. Damages can be repaired by photolyases utilizing the blue light as an energy
source. For the sake of simplicity, DNA is shown as single-stranded.
with different functions such as DNA repair factor, photoreceptor, and tran-
scriptional regulators. Collectively these genes are named as cryptochrome/
photolyase protein family (CPF). Several reviews on the CPF have been pub-
lished in the past years regarding their role in different organisms (Chaves
et al., 2006; Kavakli et al., 2017; Konig, Juhas, Jager, Kottke, & Buchel,
2017; Michael, Fribourgh, Van Gelder, & Partch, 2017; Ozturk, 2017).
Phylogenetic analyses of more than 250 photolyase/cryptochrome family
members have revealed several major classes (Fig. 2) (Asimgil & Kavakli,
2012; Kavakli et al., 2017; Ozturk, 2017; Ozturk et al., 2008). These include
Class I CPD photolyase, Class II CPD photolyase, Class III CPD photolyase,
(6-4) photolyase and single-strand specific DNA photolyases (previously
known as DASH-CRY). Class I and (6-4) photolyases with high similarity
at structural levels (Thompson & Sancar, 2002; Zhang, Wang, & Zhong,
2017) repair Pyr<>Pyr dimers and Pyr [6-4] Pyr photoproducts, respectively
(Sancar, 2003, 2016).
The (6-4) photolyases have previously been considered as only one type
and exist only in eukaryotes. However, a study with a soil born gram-negative
bacteria Agrobacterium tumefaciens indicated the presence of a new type of (6-4)
photolyase gene named PhrB while class III photolyase gene was named
PhrA. PhrB belongs to a new class named iron-sulfur bacterial cryptochromes
and photolyases (FeS-BCP) (Zhang, Scheerer, Oberpichler, Lamparter, &
Krauss, 2013). We have to note that this nomenclature of PhrA and
B should not be confused with the historical nomenclature used during early
Fig. 2 Phylogenetic tree of the cryptochrome/photolyase family. The unrooted phylo-

genetic tree of CPF member was inferred using the neighbor-joining method. The opti-
mal tree with the sum of branch lengths ¼ 10.044.
Photolyase 5
identification of photoreactivation in Escherichia coli. Two genetic loci (phrA

and phrB), which are responsible for photoreactivation, have been proposed,
however, it was shown that there is only one photolyase in E. coli (Husain &
Sancar, 1987). This nomenclature was left, and abbreviation of phr (not phrA
or B) in E. coli was exchanged with CPD photolyase. The group of FeS-BCPs
shows the largest phylogenetic distance to the other groups of the CPF. In
addition to these unusual (6-4) photolyases, another (6-4) photolyase has been
suggested to exist in Gloeobacter violaceus (Ozturk et al., 2007). Even though
FeS-BCP exists only in prokaryotes, the presence of classical (6-4) photolyase
in Gloeobacter does not allow us to classify (6-4) photolyases as prokaryotic
or eukaryotic. It might be the right time to consider (6-4) photolyases as
Class I (Eukaryotic), Class II (Fe-S BCP) and Class III (Gloeobacter (6-4)
photolyase-like).
The CRY-DASHs were found based on phylogenetic and structural
analyses of proteins found in Drosophila melanogaster, Arabidopsis thaliana,
Synechocystis and Homo sapiens (Brudler et al., 2003). CRY-DASHs were
originally thought to be a class of CRYs because of the lack of repair activ-
ity on dsDNA and failure of bacterial complementation assay in photo-
lyase deficient (phr) E. coli (Hitomi et al., 2000; Worthington, Kavakli,
Berrocal-Tito, Bondo, & Sancar, 2003). However, a subsequent study with
a CRY-DASH from Vibrio cholerae (VcCRY1) showed photolyase activity,
where VcCRY1 specifically repairs ssDNA but not dsDNA (Selby &
Sancar, 2006). It is interesting to note that multiple types of these proteins
found in algae (Asimgil & Kavakli, 2012; Chaves et al., 2011) and their
functions are reviewed in Noordally and Millar (2015).
2.1 Cofactors of the photolyases

Photolyases (PLs) are monomeric proteins with a molecular mass range from
50 to 61 kDa. They are present in bacteria, plants, fishes, birds, fungi and mar-
supial animals (Menck, 2002). PLs are associated with two chromophores
flavin adenine dinucleotide (FAD) (Fig. 3A) and methenyltetrahydro-
folate (MTHF) (Fig. 3B) or 8-hydroxy-7,8-didemethyl-5-deazariboflavin
(8-HDF) (Fig. 3C) (Sancar & Sancar, 1987). Studies with A. tumefaciens
Fe-S BCP PL revealed the presence of the new cofactors: Fe-S cluster,
and 6,7-dimethyl-8-ribityllumazine (DMRL). DMRL is an antenna chro-
mophore (Fig. 3D) (Oberpichler et al., 2011; Zhang et al., 2013). Finally,
it has been shown that Thermus thermophilus photolyase possesses flavin
Fig. 3 Structure of chromophores of photolyases. (A) Flavin adenine dinucleotide (FAD),

flavin mononucleotide (FMN), (B) methenyltetrahydrofolate (MTHF), (C) 8-hydroxy-7,8-
didemethyl-5-deazariboflavin (8-HDF), (D) 6,7-dimethyl-8-ribityllumazine (DMRL).
mononucleotide (FMN) as a second chromophore (Fig. 3A) (Ueda, Kato,

Kuramitsu, Terasawa, & Shimada, 2005).
Endogenous level of photolyase is quite low, around 15–16 molecules
per E. coli cell (Harm, Harm, & Rupert, 1968; Kavakli & Sancar, 2004).
To obtain a sufficient amount of PL for its biophysical and biochemical char-
acterization, recombinant DNA technologies were employed in E. coli cells
to over-express PL gene. Analysis of purified E. coli photolyase protein
(EcPL) indicated both apoenzyme and FAD are in 1:1 ratio (Sancar &
Sancar, 1984). FAD is strongly bound to the protein in a non-covalent man-
ner. It is the required cofactor for the catalysis of the PLs and its binding to
damaged DNA (substrate) ( Jorns, Baldwin, Sancar, & Sancar, 1987; Sancar
et al., 1987). There are four different redox states of FAD in photolyases after
the purification: oxidized (FAD), anionic semiquinone (FAD•), neutral
semiquinone (FADH•), and anionic hydroquinone (FADH). Each state
Photolyase 7
Fig. 4 Different oxidation states of the flavin adenine dinucleotide (FAD) and their
characteristic peak values in the absorption spectra. FAD accepts one electron and
one proton, both of which appear in the flavin ring system (isoalloxazine), the semi-
quinone, stable radical, FADH%, forms. When FADH% accepts one electron, fully reduced
FADH forms.
of FAD can be differentiated by absorption spectrum. Purified EcPL exhibits

different peaks: the peak at 380 nm stemming mostly from MTHF and 480,
580, and 625 nm peaks coming from blue-neutral radical form FAD (neutral
radical form of FAD: FADH%) (Fig. 4). FADH is the active form for the
DNA repair in CPD photolyases during the course of the catalysis (Kim,
Sancar, Essenmacher, & Babcock, 1993). Different oxidized forms of FAD
in the PLs can be reduced either photochemically or with dithionite
treatment.
Unlike FAD, MTHF with varying number of glutamate is loosely
attached to PL. It can easily dissociate from the PL during the purification
of the enzyme. MTHF increases the rate of DNA repair 10–100-fold rather
than affecting catalysis or substrate binding (Payne & Sancar, 1990). This
cofactor exhibits maximum light absorbance at 360 nm for the EcPL. The
interaction between the MTHF and proteins can alter the maximum absor-
bance value (Eker, Yajima, & Yasui, 1994; Sancar, 2003). The 5,10-methenyl
bridge of the MTHF is heat labile. Therefore heat can break the bridge and
MTHF cannot absorb light near-UV absorption at 360 nm. Like MTHF,
8-HDF is shown to act as photoantenna in Anacystis nidulans (AnPL) exhi-
biting an absorption maximum at 440 nm (Sancar, 2003).
In addition to FAD, the Fe-S BCP protein family has different cofactors
compared with other PLs. These are the Fe-S cluster and DMRL (Fig. 3C).
Regarding the role of [4Fe-4S] in Fe-S BCP its function is yet to be shown.
However, mutagenesis of Cys residues coordinating the Fe-S cluster
suggested that this domain is required for the stability of the Fe-S BCP
(Graf et al., 2015; Zhang, Wang, & Zhong, 2017) and Cys residues can play
a role in electron transfer (White & Dillingham, 2012). DMRL is the second
antenna shown by crystal structure and HPLC studies of the Fe-S BCP.
In fact, the I51W mutation in the A. fabrum Fe-S BCP decreased the pho-
toreduction, and the DNA repair capacity. Crystal structure of I51W mutant
Fe-S BCP showed that the DMRL binding pocket is mostly occupied by the
tryptophan residue (W51). Therefore, DMRL cannot bind to the I51W
mutant Fe-Ss BCP (Zhang et al., 2017). Hence, role of DMRL has been
proposed as to transfer energy to FAD during the course of catalysis.
2.2 The crystal structure of the photolyase

The crystal structures of the PLs from different organisms are solved via X-ray
crystallography (Fujihashi et al., 2007; Hitomi et al., 2009; Kiontke et al.,
2011; Komori et al., 2001; Park, Kim, Sancar, & Deisenhofer, 1995;
Scheerer et al., 2015; Tamada et al., 1997; Zhang, Ma, et al., 2017; Zhang
et al., 2013). Comparison of the structures revealed that PLs have two-distinct
domains connected by a loop having different amino acid residues depending
on the organismal source (Fig. 5). The first domain called the N-terminal
α/β domain consists of five β-sheets and five α-helices, which is a typical
dinucleotide-binding domain. The second domain, called C-terminal
α-helical domain, consists of 14-helices and has a cavity where the catalytic
cofactor FAD is located. The FAD is commonly present in U-shaped confor-
mation by 14 interacting amino acid residues (Sancar, 2008). The majority of
amino acids interacting with FAD are conserved at the exact same positions
Fig. 5 Superimposition of CPD photolyases of Escherichia coli, Thermus thermophilus,

Anacystis nidulans and (6-4) photolyase of Arabidopsis thaliana. Blue color indicates
the N-terminal α/β domain while orange color indicates the C-terminal α-helical domain.
Photolyase 9
between PLs with some exceptions. For example, 12 FAD interacting amino
acids are conserved between EcPL, AnPL and T. thermophilus PLs (TtPL) with
the exception of Phe-307 and Val-346 of EcPL, which are replaced by tryp-
tophan and alanine, respectively, in TtPL (Komori et al., 2001). Additionally,
the N5 atom of the isoalloxazine moiety is protonated and hydrogen bonded
to an Asn-378 residue in TtPL, which further stabilizes the FAD interaction
with apoprotein (Xu et al., 2008).
The second light harvesting MTHF cofactor is located in the cleft
between two domains of the PL. The distance from MTHF to FAD is cal-
culated as 16.8 Å in EcPL (Park et al., 1995). AnPL has an additional space in
the interior part between the clefts where it accommodates MTHF (Tamada
et al., 1997). PLs specifically bind either to ssDNA or dsDNA containing
Pyr <>Pyr damage with the specific binding constant (Kd) of 109 M
(Husain & Sancar, 1987). Analysis of the solvent accessible surface of EcPL
indicates that FAD can interact Pyr<>Pyr through the hole (Fig. 6A) (Park
et al., 1995). Surrounding regions of the hole are rich with positively
charged amino acid residues indicated by the blue color. In fact replacement
of the amino acids in the blue region results in the change in the affinity of
the EcPL to the substrate (Baer & Sancar, 1993).
The crystal structures of the ssDNA repair enzymes of A. thaliana
Cryptochrome 3 (AtCRY3) and Synechocystis CRY (also called as CRY-
DASHs) have also been determined (Brudler et al., 2003; Huang et al.,
2006; Klar, Pokorny, Moldt, Batschauer, & Essen, 2007). Analysis showed
that the overall structures of the CRY DASHs are very similar to class I EcPL
with some differences. One main difference is that AtCRY3 DNA binding
region is more polar compared to class I PLs. Although the substrate-binding
regions of both classes of enzymes are similar, CRY-DASH has a weak affin-
ity toward damaged or undamaged dsDNA (Kizilel et al., 2012).
The crystal structure of Fe-S BCP from A. tumefaciens is solved with a
resolution of 1.45 Å (Zhang et al., 2013). The comparison of this structure
with other PLs revealed a very similar 3D shape with root-mean-square dif-
ference (RMSD) values range from 2.8 to 3.5 Å. The FAD is present in a
U-shaped conformation and stabilized by hydrogen bonding interactions
with the side chain of Arg369 and the carbonyl group of Tyr391 in Fe-S
BCP. The analysis of the second chromophore-binding site suggested that
only DMRL could fit with the electron density map and its presence is con-
firmed by HPLC. Unlike EcPL DNA binding region, where one side has
hydrophobic residues and other side has polar residues, the Fe-S BCP sub-
strate binding site is very similar to (6-4) A. thaliana PL (AtPL) having
His-His-X-X-Arg motif (Hitomi et al., 2001). The replacement of the
Fig. 6 (A) Surface contour image indicates FAD binding region and surface exposed
amino acids of E. coli photolyase (PDB ID: 1DNP). Blue color represents amino acids with
basic groups while red color represents amino acids with acidic groups. Carbon atoms of
FAD are shown in orange. (B) (6-4) photolyase from Agrobacterium tumefaciens (PDB ID:
4DJA) and cofactor binding pockets. There are 4-cysteines interacting with the 4Fe-4S
cluster. Fe atoms are shown in orange and sulfur atoms in yellow.
conserved His366 in FeS-BCP results in loss of its DNA repair activity (Graf
et al., 2015). This is consistent with previous studies where it is shown this
particular amino acid is required for DNA repair in (6-4) PLs. The FeS-BCP
possesses a [4Fe-4S] cluster in which four iron atoms are coordinated by four
Cys residues (Cys-350, Cys 438, Cys441, and Cys454 in A. tumefaciens)
(Fig. 6B).
2.3 Reaction mechanism of DNA repair mediated by CPD

photolyase
The reaction mechanism of the Class I CPD photolyase has been revealed
using EcPL (Sancar, 2003, 2008, 2016). Photolyase binds to the backbone of
Photolyase 11
the damaged DNA strand by ionic interactions. This binding occurs around
the cyclobutane dimer in a light-independent manner. Upon binding, PL
flips the dimer out into the active site cavity to make contact with the flavin
(Mees et al., 2004; Park et al., 1995) which leads to stable enzyme-substrate
complex ( Jordan, Alderfer, Chanderkar, & Jorns, 1989; Jorns, Sancar, &
Sancar, 1985). Exposure of this complex to light initiates catalysis; the
FADH and MTHF photoantenna absorb a photon and MTHF transfers
the excitation energy to fully reduce FADH by F€ orster dipole-dipole res-
onance energy transfer to increase the efficiency of the overall DNA repair.
Light absorbance generates excited FADH• which donates an electron to
the Pyr <>Pyr, where it cleaves the C5–C50 and C6–C60 bonds of the
cyclobutane ring and generates free thymine bases. The enzyme and the
product dissociate. The electron is transferred back into FADH• to regen-
erate FADH, ready for the next round of catalysis (Fig. 7). Photolyase
repairs damaged DNA using a mechanism of light-induced electron transfer
that does not result in a net change of the redox state of the enzyme (Sancar,
2016). This repair is achieved with a high quantum yield of 0.84.
Fig. 7 Reaction mechanism of E. coli CPD photolyase. First, methenyltetrahydrofolate

absorbs a photon from the blue light wavelengths and transfers the energy to flavin.
The excited electron from Flavin (FAD) breaks the cyclobutane ring and is transferred
back to flavin.
To understand how this is achieved Tan et al. performed femtosecond spec-

troscopy using EcPL (Tan et al., 2015). Their results revealed the presence of
the cyclic electron tunneling mechanism. Unique bended structure of FAD
within the EcPL where adenine and lumi-flavin moieties of FAD come very
close which allows fast direct intramolecular electron transfer from lumi-
flavin moiety of four redox states of FAD to the substrate of Pyr <>Pyr
in 250 ps through direct tunneling (Liu et al., 2011, 2013).
Studies with EcPL (Tan et al., 2014) and ssDNA repair enzyme of
V. cholerae CRY1 (Saxena, Wang, Kavakli, Sancar, & Zhong, 2005) showed
the rate of resonance energy transfer (RET) between the MTHF and
FADH in PLs depends on the distance between chromophores and their
orientations within the enzyme. The RET time from MTHF* was calcu-
lated to be 170 ps for EcPL, where the distance between the two chromo-
phores is exactly 16.8 Å. The RET time for VcCry1 where the distance
between MTHF and FADH is predicted as 15.2 Å was calculated to be
60 ps (Zhang, Wang, & Zhong, 2017).
During the purification of the EcPL, where it is exposed to oxygen, the
FAD is found in neutral semiquinone form (FADH•) ( Jorns, Sancar, &
Sancar, 1984). Enzyme-FADH• is inactive unless it is treated with blue-
light. Then energy is transferred to FADH• by nearby Trp residue within
the EcPL. Trp306 is identified as a primary electron donor during the pho-
toreduction of FAD (Li, Heelis, & Sancar, 1991). Then, in vitro studies pro-
posed two electron transfer mechanisms from Trp306 to FADH•; electron
hops “Trp triad,” and electron travel through α–helix side chain of Phe 366.
To understand whether such mechanism is part of the EcPL photocycle dur-
ing the DNA repair in vivo mutant phr genes (Trp306Phe and Trp382Phe)
are expressed at endogenous level in phr deficient E. coli. Results indicated
that neither proposed electron transfer pathways are a part of the photocycle
of EcPL in vivo (Kavakli & Sancar, 2004).
2.4 Reaction mechanisms of DNA repair mediated by (6-4)

and other classes of the photolyases
Exposure of the DNA to UV irradiation for a long time not only generates
Pyr <>Pyr but also Pyr [6-4] Pyr photoproducts. Unlike Pyr <>Pyr pho-
toproducts, Pyr [6-4] Pyr photoproducts are formed from the pyrimidine
excited singlet state. Initially, Pyr [6-4] Pyr photoproduct is thought to
be repaired by the excision repair system. An enzyme called (6-4) photo-
lyase, which has the ability to directly repair Pyr [6-4] Pyr, was discovered
in Drosophila (Todo et al., 1993). Following that (6-4) photolyases were
Photolyase 13
discovered in some other eukaryotic organisms. Therefore it is thought

that this type of repair is only found in eukaryotic organisms. Recently, a
new class has been discovered in more than 350 bacterial organisms
(Oberpichler et al., 2011) and shown to possess Pyr [6-4] Pyr repair activity
(Zhang et al., 2013). Despite the fact that (6-4) photolyases are very similar
to CPD photolyases at the structural level and having a similar cofactor
contents (Kim, Malhotra, Taylor, & Sancar, 1996; Ozturk, 2017; Ozturk
et al., 2008; Schelvis & Gindt, 2017; Zhao et al., 1997), biochemical char-
acterization of this enzyme revealed that (6-4) photolyases belong to differ-
ent classes. Therefore, it is expected that class II, III, and (6-4) photolyases
would have the same reaction mechanism as Class I photolyase. However,
(6-4) photolyases must first thermally convert the open form of the (6-4)
photoproducts to an oxetane intermediate before the repair which is the
main difference to the CPD PLs. In contrast to CPD photoproducts, in
which breaking of the UV light-induced bonds restores the bases to their
canonical forms, the breaking of the C-5–OH and C6–C4 bonds of the
(6-4) photoproducts would not repair DNA but would, in fact, generate
two damaged bases. Therefore, formally an enzyme that repairs the (6-4)
photoproduct must catalyze both bond breakage (lyase) and group transfer
(transferase) reactions. Surprisingly, the (6-4) photolyase accomplishes this
difficult task, even though it occurs with a rather low quantum yield
(Hitomi et al., 1997). Several repair models were proposed for the (6-4)
photolyase: (i) oxetane intermediated formation in the ground state before
photochemical reaction (Hitomi et al., 2001), (ii) the primary photochem-
ical reaction followed by water molecule formation to split bond breakage
(Maul et al., 2008) and (iii) a two-photon repair mechanism (Yamamoto,
Plaza, & Brettel, 2017). In any repair models, the involvement of two His
residues, which are His354 and His358 from Xenopus laevis (6-4) PL
(XlPL), play critical roles (Hitomi et al., 2001) (His365 and His369 in
D. melanogaster PL, DmPL; His364 and His368 in A. thaliana). The replace-
ment of these amino acids results in significant activity loss of the Xl (6-4)
PL although the affinity of the mutants is comparable with the wild-type
enzyme. In fact, X-ray crystallographic study indicates His354 (His365
in DmPL) contacts with the OH group of C5 side of the 50 (Fig. 8A)
(Maul et al., 2008). The proton is transferred from the His354 (6-4) pho-
toproducts to generate protonated neutral radical photoproduct for effi-
cient DNA repair.
By using the ultrafast spectroscopy Li et al. (2010) showed the steps of the
catalysis in At (6-4) PL. They revealed that the repair is a cyclic proton
Fig. 8 (A) Active site of the Drosophila melanogaster (6-4) photolyase (PDB ID: 3CVU)
with (6-4) photoproduct. During the course of catalysis, His365 donates a proton to
repair (6-4) photoproduct. The red dotted line indicates the distance between the
His residues to FAD in Å. (B) The photocycle DNA repair by (6-4) photolyase. In the very
first step electron ejection occurs from substrate I to II in 280 ps by direct electron
tunneling from lumiflavin (LfH) to substrate via forward electron transfer (FET2). Then,
a proton is transferred from His364 to substrate generating substrate III in 481 ps which
is branched by the back electron transfer (BET2) occurring in 57 ps without any repair
(from substrate II to substrate I). During the course of repair, several rearrangements
occur within the substrate (from III to IV). Finally, the proton with an electron goes back
to His364 and generates two free thymine bases, which takes more than 10 ns.
Photolyase 15
transfer between the enzyme and the substrate, where a single electron
completely repairs the (6-4) photoproduct in its ground state. As shown
in Fig. 8B, once photoinduced charge separation occurs between the
FAD and the substrates, the reactions proceed in different pathways. These
pathways have different backward and forward energies, which causes a
quantum yield of DNA repair comparable to CPD photolyase. Addition-
ally, they also showed proton transfer (His354 in Xl (6-4) PL) is the rate-
limiting step in the catalysis of the (6-4) PL. It is expected that Class III and
ssDNA photolyases would have the same reaction mechanism as Class
I repair photolyases.
3. Conclusion
Phylogenetic analyses of cryptochrome/photolyase protein family
have been revealed for different classes of the photolyases. Although homol-
ogy of PLs is quite low at the primary structures level, the comparisons of the
crystal structures of the PLs reveal remarkable similarity among different
classes with some differences. Studies with enzyme–substrate co-crystal
structures of different PLs and the identification of reaction intermediates
by fast spectroscopic methods enabled us to pinpoint reaction mechanism
during the course of DNA repair. Recent studies highlighted that CPD
and (6-4) photolyases use a very similar energy transfer strategy with a cyclic
electron transfer radical mechanism consistent with the in vivo studies.
Exceptionally, a critical proton donation from His residue is needed to trans-
fer oxygen atoms between two damaged bases during the course of DNA
repair mediated by (6-4) photolyases. Photolyase still remains an excellent
system to study intra-protein energy transfer with multiple tunneling or
hopping pathways.
Acknowledgment
We would like to thank Dr. Mehmet Tardu for helping us to generate Fig. 2. We also like to
thank Anna Elms for her critical reading of the manuscript.
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CHAPTER TWO
TFIIH: A multi-subunit complex

at the cross-roads of
transcription and DNA repair
Olga Kolesnikovaa,b,c,d, Laura Radua,b,c,d, Arnaud Poterszmana,b,c,d,*
a
Institut de Genetique et de Biologie Moleculaire et Cellulaire Illkirch Cedex, C.U. Strasbourg, France
b
Centre National de la Recherche Scientifique, UMR7104, Illkirch, France
c
Institut National de la Sante et de la Recherche Medicale, U1258, Illkirch, France
d
Universite de Strasbourg, Illkirch, France
*Corresponding author: e-mail address: arnaud.poterszman@igbmc.fr
Contents
1. Introduction 22
2. Composition, topology and structure of TFIIH 25
2.1 TFIIH composition: 10 subunits and more 25
2.2 Topology and structure of TFIIH 28
3. The XPB translocase and the XPD helicase 36
3.1 XPB: A translocase rather than a helicase 37
3.2 XPD a bona fide helicase: Damage recognition and DNA opening 38
4. Regulatory core-TFIIH subunits 39
4.1 The p34/p44 pair: Regulation of XPD helicase 39
4.2 p52 and p8: Modulation of XPB activity 41
4.3 p62: An anchor for partners 41
5. Transcription regulation: The role of TFIIH 42
5.1 Promoter opening 43
5.2 Promoter escape 43
6. TFIIH in nucleotide excision repair 46
6.1 XPC-mediated detection of DNA lesions and recruitment of TFIIH 46
6.2 TFIIH opens DNA and verifies the damage 47
6.3 TFIIH-mediated enrollment of the XPG endonuclease 50
7. TFIIH, human diseases and next generation cancer therapies 51
7.1 Molecular basis of XP, CS and TTD: Insights into TFIIH regulation 51
7.2 Viral pathogenesis 52
7.3 TFIIH as a therapeutic target: Small molecule inhibitors 53
8. Conclusions 54
Acknowledgments 55
References 55
#
Advances in Protein Chemistry and Structural Biology, Volume 115 2019 Elsevier Inc. 21
ISSN 1876-1623 All rights reserved.
https://doi.org/10.1016/bs.apcsb.2019.01.003
22 Olga Kolesnikova et al.
Abstract
Transcription factor IIH (TFIIH) is a multiprotein complex involved in both eukaryotic
transcription and DNA repair, revealing a tight connection between these two pro-
cesses. Composed of 10 subunits, it can be resolved into a 7-subunits core complex with
the XPB translocase and the XPD helicase, and the 3-subunits kinase complex CAK,
which also exists as a free complex with a distinct function. Initially identified as basal
transcription factor, TFIIH also participates in transcription regulation and plays a key role
in nucleotide excision repair (NER) for opening DNA at damaged sites, lesion verification
and recruitment of additional repair factors. Our understanding of TFIIH function in
eukaryotic cells has greatly benefited from studies of the genetic rare diseases
xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy
(TTD), that are not only characterized by cancer and aging predispositions but also
by neurological and developmental defects. Although much remains unknown about
TFIIH function, significant progresses have been done regarding the structure of the
complex, the functions of its catalytic subunits and the multiple roles of the regulatory
core-TFIIH subunits. This review provides a non-exhaustive survey of key discoveries on
the structure and function of this pivotal factor, which can be considered as a promising
target for therapeutic strategies.
1. Introduction
Multiple genome-caretaking mechanisms counteract the deleterious
effects of DNA damage and prevent toxicity, mutagenesis and genomic instabil-
ity. The nucleotide excision repair (NER) pathway discovered in the early
1960th (Hanawalt & Setlow, 1960) corrects by a “cut-and-patch”-type reaction
different structurally unrelated DNA lesions raging from pyrimidine–pyrimidine
intra-strand cross links, induced by UV light to bulky DNA adducts induced
by environmental carcinogens, reactive oxygen species and cellular metabolites.
This process is conserved across evolution but the proteins involved differ
in prokaryotes and eukaryotes. In bacteria, during global genome NER
(GG-NER), damage is detected by UvrA acting in concert with UvrB, which
scans DNA for damage recognition. Alternatively, if the damage is first encoun-
tered by a stalled RNA polymerase (RNAP), the transcriptional repair coupling
factor (TRCF), also known as Mfd, pushes RNAP off from the lesion and
recruits UvrA2 to the damaged site. In both cases, after damage recognition,
UvrA dissociates from the pre-incision complex and the UvrC dual nuclease
incises the damaged strand on both the 30 and 50 sides of the damage. Finally,
UvrD removes the incised oligonucleotide and UvrC, while UvrB remains
bound to the gapped DNA until polymerase I fills the gap (Kisker, Kuper, &
Van Houten, 2013).
TFIIH: A multi-subunit complex 23
In eukaryotes, NER requires the concerted action of more than 20 fac-

tors that sequentially recognize a lesion, excise it in the form of an oligonu-
cleotide containing the damage, and fill in the resulting gap by repair
synthesis (Fig. 1) (Marteijn, Lans, Vermeulen, & Hoeijmakers, 2014). As
in bacteria, two NER pathways have been identified. In GG-NER, dam-
aged DNA not undergoing transcription is repaired. This pathway is initi-
ated by protein complexes UV-DDB and XPC-HR23B which specifically
detect and bind to damage-containing DNA. Transcription-Coupled NER
(TC-NER) corrects damages in actively transcribed regions. When RNA
polymerase II engaged in transcription elongation encounters the lesion on
the transcribed DNA strand of actively transcribed genes (Mellon, Spivak, &
Hanawalt, 1987) it stalls. The stalled polymerase interacts with TC-NER
initiation factors, which include the CSA and CSB proteins, as well as
the UV-specific scaffold protein A (UVSSA) and the ubiquitin-specific pro-
tease 7 (USP7). This probably results in reverse translocation (backtracking)
of the TC-NER-initiation complex, rendering the DNA damage accessible
for repair.
For both pathways, following damage recognition, the XPB and XPD
ATPase/helicases within the multi-subunit transcription/DNA repair factor
TFIIH unwind the DNA around the damage. The opened state of the DNA
is then stabilized through the recruitment of additional factors such as XPA,
replication protein A (RPA) and the endonuclease XPG. The recruitment of
the endonuclease ERCC1-XPF to the opened DNA structure completes
the formation of the pre-incision complex and triggers dual incision and
removal of the damaged oligonucleotide. The single-stranded gap is subse-
quently filled by DNA repair synthesis.
Three severe genetic disorders result from mutations in NER genes,
namely xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and
Cockayne syndrome (CS). Cell fusion studies identified multiple comple-
mentation groups, each being found to be associated with mutations in
individual genes. Eight complementation groups were identified for XP
(XP-A to XP-G and variant), five for CS (CSA, CSB, XPB, XPD, and
XPG) and three for TTD (XP-B, XP-D, and TTD-A). These syndromes
are genetically complex and display diverse phenotypes from mild solar
sensitivity to skin cancer, neurological degeneration and developmental
disorders: XP being a highly cancer-prone skin disorder, whereas CS and
TTD are cancer-free multisystem diseases.
TFIIH was the first example of factor with established roles in both tran-
scription initiation and NER, but is not an exception. Indeed, other NER
factors also contribute, in addition to DNA repair, to the regulation of gene
24 Olga Kolesnikova et al.
Fig. 1 See legend on opposite page.

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At the outset I was more fortunate than on the previous day, for
when I had gotten up close to them I found in front of me cows and
calves, young things of one or two years old. Singling out a fat young
cow, distinguished by her glossy coat of hair, I forced my horse right
up against her and brought her down at the second shot. I pulled
rein, stopping my horse as suddenly as was possible at the
breakneck speed at which he was going, and in another moment the
herd had spread out, and I was completely surrounded by the
rushing mass of animals which my attack had set in motion.
The air was so clouded with dust that I could hardly see more
than twenty yards from where I was standing, near the carcass of the
cow I had killed. There was danger of being run over by them, but
they separated as they approached, passing on either side of me, a
few yards distant. After a while the rushing crowd thinned, and up
rode Captain Chiles exclaiming: “Why don’t you kill another?”
Fifty yards from us they were rushing by, all in the same
direction. I again dashed into the midst of them, pressing my horse in
pursuit of another young cow. She shot ahead of everything,
increasing her speed so that I could hardly keep sight of her. While
thus running at full speed my horse struck a calf with his breast,
knocking the calf down flat, and almost throwing himself also. I
pulled up as quickly as possible, turned around and shot the
prostrate calf before it could get up. So I had two dead in, say twenty
minutes. After this day’s experience I had no trouble in killing all the
buffalo we needed for our own consumption. For a week or ten days
they were hardly out of sight. We found them as far west as Pawnee
Rock. All told, I killed about twenty on the journey out and back. A
good steak, cut from the loin of a buffalo cow, broiled on the coals
with a thin slice of bacon attached to it to improve its flavor, was
“good eating,” and I soon became an accomplished broiler.
IV.
Companions of Voyage.
Before reaching Pawnee Rock we overtook a train of thirty

wagons belonging to the leading freighters of the West, Majors,
Russell & Waddell, with which we traveled to Fort Union, their freight
being consigned to that post. This train had thirty wagons, built, I
believe, in Philadelphia, with heavy iron axles and spindles, which
seemed superior to any others I had seen on the prairies. Hagan
was wagonmaster and Hines his assistant. The former was a sandy-
haired man, who rode a large bay mule, a drowsy animal with
immense lop ears that moved back and forth as he walked. This
ungainly mule, I found out, in a day or two afterwards, had his good
points. He could run as fast and get up as close to a buffalo as any
horse in either outfit.
Notwithstanding Hagan’s generally uncouth appearance, he was
a man of sterling worth and a capital hand at killing buffalo.
Subsequently we joined in many chases, and I found him an
agreeable companion. On the rear end of each of the wagons in
Hagan’s train there was pasted a set of printed rules for the
government of the employees in the service of Majors, Russell &
Waddell. Both liquor and profanity were absolutely prohibited, but of
the strict enforcement of the rules I cannot speak.
While riding in advance of the train, in company with Captain
Chiles, we saw our Mexican friend, whose acquaintance we had
formed at Westport, the master of his own train, galloping toward us,
with a buffalo cow following close behind his horse. As was his habit,
he had attacked the animal with his spear, stabbing her until she
became infuriated so that she turned on him and was following him;
it occurred to me she was pressing him a little too closely to be
agreeable. We rode rapidly toward him, and as we were drawing
near the cow became so exhausted by loss of blood that she
stopped still, when Captain Chiles rode up and gave her a broadside
with his shotgun, which finished her.
Whenever they found buffalo in plenty the Mexicans would halt
for several days and kill enough to supply their trainmen. They
preserved the meat by cutting it into thin strips and hanging it on
ropes about the corral until it was dried by the sun. But thus cured, it
had a sour and disagreeable taste to me. The Mexicans would stew
it with quantities of red pepper and devour it with great relish.
As we approached the valley of the Little Arkansas, where the
view of the country was more extensive than any we had yet seen,
there was no limit to the herds of buffalo, the face of the earth being
covered with them. We camped at noon at the crossing of this
stream. The buffalo were crossing the creek above us, moving
westward, in bands of from twenty-five to a hundred or more. At the
crossing they had a trail cut down through the steep banks of the
stream three or four feet in depth.
But I had had enough of buffalo chasing, except when we were
in need of fresh meat. It was too much like riding out into the pasture
and killing your own domestic cattle. I found antelope hunting much
better sport.
After Walnut creek, the next place of interest was Pawnee Rock
near which many battles between the traders and the Indians had
taken place. This bluff, facing the road on the right hand side, at a
distance, perhaps, of a hundred yards, was of brown sandstone
about fifty feet high, the bluff end of the ridge extending down to the
river bottom. I climbed up the almost perpendicular face of the
elevation, where I found many names cut in the soft stone—names
of Santa Fé traders who had traveled the trail, among them that of
Colonel M. M. Marmaduke, who crossed to Mexico as early as 1826,
and was afterwards governor of Missouri, and James H. Lucas, a
prominent and wealthy citizen of St. Louis.
We were not particularly apprehensive of Indian troubles,
although we knew the Cheyennes were turbulent. Elijah Chiles, a
brother of our captain, had been loading goods at Kansas City when
we left—a train of twenty-six wagons for the Kiowas and Comanches
—and was doubtless a few days’ drive behind us. But we kept on the
lookout day and night; the guard around the cattle was doubled, and
each teamster had a gun of some sort, which he kept strapped to the
wagon bed, loaded and ready for service.
V.
Pestiferous Indians.
All the while we knew the Indians could wipe us out if they were
determined to do so. In both trains there were not above sixty men,
while there were, nearby, warriors by thousands.
A day’s journey beyond Pawnee Rock, we were visited by a
hunting party of fifteen or twenty young Kiowa bucks, the first real
“wild” Indians we had seen. They did not seem the least wild, however,
but uncomfortably “tame,” and disposed to get very familiar on short
acquaintance. They were evidently out on a lark, and disposed to
make us the objects of their amusement that afternoon.
They scattered up and down the length of both trains, talking and
laughing with the teamsters. Two of them took particular fancy to my
friend Reece, riding on either side of him, taking hold of his arms and
seeming to admire his long hair and the handsome horse he rode.
Reece was not at all afraid of them and permitted no undue
interference with his person or property.
Reece was no coward. While we were still in the dangerous region,
he would ride for miles ahead of the train, alone, dismount and lie
down to rest or sleep. When I said to him that he was incurring
unnecessary risk of being killed by the Indians, he remarked that if
they did kill him they could not rob him of much in this world.
Along where we were traveling at the time of the visit of the Kiowa
bucks, the river bottom was as smooth as a billiard table. Hagan’s train
was in the lead of ours a space of perhaps thirty yards intervening.
Hagan and I were riding abreast at the rear of his train, when suddenly,
two of the young bucks raised up a loud whoop and started their
horses at full speed. Taking a corner of their blankets in each hand and
holding them above their heads so that they made a flapping sound in
the air, they went sweeping along right against the cattle, almost
instantly creating a stampede, the cattle turning out of the highway
making the big wagons rattle as they went.
For an instant Hagan sat on his mule stock still, apparently
dumbfounded. In another moment he put spurs to his mule, intending
to head the fleeing cattle. But instead of running, the mule suddenly
“bucked,” throwing Hagan and his saddle also (the girth breaking) over
his head and landing him in the road, flat on his back. Hagan got up,
pulled himself together and rubbed the dust out of his eyes, but said
nothing, though gifted in the way of eloquent profanity.
No great harm resulted from the stampede. Some others of the
party of Indians ran ahead and stopped the cattle. There was no
collision of wagons and no damage, but the affair left an ugly feeling of
resentment among the teamsters toward the Indians. The Indians
laughed and talked about the affair among themselves. Any effort to
punish them was out of the question, the entire tribes of Kiowas and
Comanches being encamped within a day’s journey above us.
THE MULE SUDDENLY BUCKED.
The Indians kept along with the train all of the afternoon.
Observing my horse and accoutrements, they inquired through Juan,
the Spaniard, if he was fleet and good for buffalo, and pressed me to
go out with them for buffalo the next day. I would gladly have seen the
Indians engaged in a buffalo chase, but declined the invitation, making
such excuses as I could without expressing any want of confidence as
to their good fellowship. My scalp was intact and I felt disposed to keep
it so.
The Kiowas begged Captain Chiles and Hagan to give them some
flour and sugar, but they refused, knowing that a donation would be
necessary later on, when we should meet the entire tribes of Kiowas
and Comanches encamped above us, awaiting the arrival of their
agent and the train load of goods for them.
Late in the evening, after we had corralled and the cooks were
preparing to get supper these Indians having ridden off in the direction
of the river, two of them reappeared. They returned to the camp, each
with a bundle of dry driftwood, picked up on the river bank, which they
threw down near the camp fire. This meant that they wanted supper,
and Captain Chiles gave directions for the preparation of food for
them. The Indians took supper with us, after which they departed,
evidently feeling better and good naturedly disposed toward us.
That night there was much discussion of the Indian problem, with
which we seemed now confronted. At noon the next day, as the cattle
were being driven into the corral, another party of young warriors made
their appearance at our camp, and came near involving us in a serious
conflict. The trouble was brought on by the impatient action of our
assistant wagonmaster, Rice. Four or five young fellows rode up into
the rear entrance of our corral and were sitting there on their horses
looking on at the yoking of the cattle. They partially blocked up the
opening and interfered with egress of the teams. Rice, coming up
behind them, without warning gave one of their horses a blow with a
heavy blacksnake whip. The horse sprang forward, nearly unseating
the rider, who, as soon as he could gather up the reins of his bridle,
turned upon Rice in a towering rage, jerked an arrow from its quiver
and fixed it in his bow. Forcing his horse right upon Rice, the Indian
punched him with the point of the arrow until he knocked his hat off his
head. Rice made no effort to resist the affront and threatened assault,
but kept backing out of the Indian’s reach.
I was standing near by and seized my pistol, thinking that a fight
was imminent. At the height of the excitement, Captain Chiles made
his appearance and commanded peace, in manner and language that
the Indians could understand, but it required some time and a deal of
talk to get them quieted. They denounced Rice’s conduct as an insult
they were bound to resent, and declared they would kill Rice sooner or
later. Captain Chiles, speaking through Juan, our Spaniard, told them
that if they commenced killing they would have to kill us all, for we
were bound to stand together when it came to that. After a long
wrangle the Indian said he would be satisfied if allowed to give Rice a
sound flogging with a whip, but Captain Chiles refused. Finally the
Indians seemed to recover their composure, to some extent, and rode
off in the direction of the main camp.
* * * * *
Somewhere thereabout, in the river bottoms, I saw the ruins of an
old adobe fort. “Old Fort Atkinson,” doubtless named for and
established and built by the command of Colonel Henry Atkinson of the
regular army, with whose military career I happened to be somewhat
familiar. The remains of the old fort excited my interest, but I do not
recollect to have seen the place mentioned by any of the numerous
accounts that have been written of the Santa Fé trail.
PUNCHED HIM WITH THE POINT.
The fort was probably built in 1829. At that time a body of regular
troops was sent out on the trail as a protection to the traders. Colonel
Henry Atkinson was ordered west in 1818 and placed in command of
the Ninth Military department, then comprising the entire country west
of St. Louis, as well as Illinois and Wisconsin, with headquarters at
Fort Bellefontaine, near St. Louis. He was soon afterward advanced in
rank to brigadier general and held the command at Jefferson barracks
until his death in 1842. The military post at Council Bluffs, Ia., was
established by Colonel Atkinson in 1819, when he and his troops were
transported on the first steamboats ascending the Missouri river. He
served with distinction in the Black Hawk War, in command of the
forces.
VI.
At the Kiowa Camp.
The train had got under way the next morning when the lodges
of the Kiowas loomed up in sight of us. The camp seemed to extend
over territory a mile square. The Indians said the entire tribe was
assembled there—chiefs, warriors, squaws and papooses. Presently
we could see them moving towards us, hundreds of them, on
horseback and on foot, all sorts and sizes, men, women and
children, coming to take a view of the white man and his belongings
as they passed.
Soon we could see also the lodges of the Comanches,
appearing about equal in number, and covering a like extent of
country. The two camps were a mile or more apart.
It had been agreed between the wagonmasters that we would
not make the usual noonday halt that day, but would drive by the
Indian camps and as far beyond as it was possible for the cattle to
stand the travel. We had anticipated a great throng of Indians, and
here they came by the hundreds!
Some of the “big men” among them had guns or pistols, but the
greater number, in fact almost every one, had a bow and quiver of
arrows slung over his shoulders, even the children who looked not
over ten years old. One chief wore a complete outfit of blue, with the
insignia of a captain of the United States army, and had a Colt’s
revolver, but nearly all of them were naked to the waist, with a
breech-clout and a sort of kilt of buckskin around the loins, hanging
down nearly to the knees. Some wore moccasins, while many were
barefooted.
The little fellows, nude, save for a breech-clout, had little bows
about a foot long, with arrows of cactus thorn, with which they would
shoot grasshoppers and other insects, showing astonishing skill.
Numbers of the warriors carried spears, with long handles, glittering
in the sunlight as they rode along, giving the caravan the
appearance of a vast army of Crusaders on the march to the Holy
Land.
Captain Chiles, endeavoring to shift the responsibility and
escape the annoyance of the Indians, pointed to Reece, on his fine
horse, and said: “There is the captain; talk to him. Ask him for what
you want.” But they could not be so easily deceived. It is said that
you cannot fool Indians in this particular; that they never fail to
distinguish the wagonmaster, and appear to select the chief of any
crowd or caravan intuitively.
As we were traveling along the Indians gave frequent exhibitions
of the speed of their horses, running races with each other, but at a
sufficient distance not to frighten or stampede our cattle. The
younger men kept up a continual chattering and laughing; horse
racing seemed their great amusement. The young fellows of the visit
renewed their invitation, urging me to join them in a buffalo chase,
explaining that the herds were not far off, and expressing a great
desire to see a trial of my buffalo horse in a chase with theirs. I again
declined. The train was continually moving and would not be stopped
to suit my convenience, and there were other reasons, not
unreasonably discreet.
The head men of the tribes, addressing the wagonmasters,
complained that they were in great need of supplies, owing to the
delay in the arrival of their annuities, and asked a gift from the two
trains. The two wagonmasters, after some demurring, proposed to
them that if they, with all their people, would withdraw from, and
cease to follow the train, and desist from annoying us, after we had
corralled, we would go into camp and give them such supplies as we
could spare.
To this proposition the chiefs agreed. One of the leaders began
talking in a loud voice to the multitude, gradually riding off from us,
the crowd following. Reaching a knoll which elevated him so that he
could overlook them, he dismounted and proceeded to make a
speech. They seemed a little slow about leaving, the multitude
appearing to be not altogether governed by the leaders, but nearly
all finally withdrew in the direction of their own camp. Driving on a
few hundred yards further, our corrals were formed and the cattle
were driven off some distance for water, while preparations were
made for cooking dinner.
In a little while the chiefs, representing both tribes, made their
appearance at our corral, where the wagonmasters of both trains
had met to hold the diplomatic conference to determine how much of
a gift of supplies they were expecting from us.
The Indian chiefs dismounted from their horses, walked into the
corral and sat down on the ground, in the semi-circle, to the number
of perhaps a dozen and were soon joined by the wagonmasters,
together with our interpreter Juan.
Writing now, in the year 1901, solely from memory, forty-three
years since this scene occurred, I am unable to recollect all that was
said, or the names of any of the Indians who were present and took
part in this parley. No doubt San Tanta, that famous Kiowa chief, was
among them, but I took no notes whatever of this journey, and am
forced now to rely entirely on my memory. I recall that it was stated
that one of the most influential of the Comanche chiefs who was
there was an out-and-out Spaniard or Mexican, speaking the Indian
language as well as anybody, and was generally known and
recognized as among the meanest, most cruel and blood thirsty of
the Comanche tribe. One of the elder looking Indians produced a big
pipe, filled it with tobacco, lighted it, and after taking a few puffs
himself passed it to the one next to him. Thus the pipe was passed
around to each one in the circle until all had taken part in the smoke.
The Indians were dignified, discreet and cautious, as appeared to
me during the conference, leaving the impression that our troubles
with them were about to terminate, and this proved to be the fact.
At the close, and as a result of the council, a half-dozen sacks of
flour, half that many sacks of sugar, and a lot of sides of bacon were
brought forth from the mess wagons and stacked up on the ground,
near where the collection of dignitaries of the prairies were sitting,
smoking the pipe of peace and good fellowship.
I thought the Indians regarded the things we were giving them,
as a sort of tribute we were under obligations to pay for the privilege
of passing through their country unmolested.
Pack mules were brought up, the supplies were loaded on them
and they departed in the direction of the general camp, those
engaged in the conference soon following.
In the evening, before we broke camp, two young bucks came
galloping into the camp. Addressing Captain Chiles, they said that by
instruction of their chief they had come to return a pair of blankets
that had been stolen by one of the tribe. They threw down the
blankets and the captain called to the men at the mess wagon to
give them a cup of sugar each, saying that it was the first instance in
his life when an Indian had restored stolen property.
VII.
To the Cimarron.
Escaping any further delay from Indians or from other causes,

good headway was made by the trains up the Arkansas until we
reached the “lower crossing.” It had been determined by the
wagonmasters that we would cross the river here, taking the
Cimarron route. Although the river was fordable, yet it was quite
tedious and difficult to get the heavily loaded wagons across the
stream, the water being waist-deep and the bottom uneven.
Neither an ox nor a mule will pull when he gets into water
touching his body. The mule, under such circumstances, always has
a tendency to fall down, and so get drowned, by becoming entangled
in the harness. To meet this emergency the ox teams were doubled,
ten yoke being hitched to each wagon, and were urged to do their
duty by a half-dozen drivers on each side, wading through the water
beside them.
The greater part of one day was taken up in getting the wagons
across, but it was accomplished without serious loss. Everything
being over, we encamped at the foot of the hill on the opposite side,
and rested a day, in recognition of the Fourth of July. We fired some
shots, and Captain Chiles brought forth from his trunk some jars of
gooseberries, directing the cooks to make some pies, as an
additional recognition of the national holiday. The gooseberries were
all right, but the pie crust would have given an ostrich a case of
indigestion.
The old Santa Fé trail, from the lower crossing of the Arkansas,
ran southwest to the Cimarron, across a stretch of country where
there was no water for a distance of nearly sixty miles, if my memory
serves me correctly. All the water casks were filled from the
Arkansas river for the use of the men, but of course there was no
means of carrying water for horse or ox.
The weather was warm and dry, and now we were about to enter
upon the “hornada,” the Spanish word for “dry stretch.” Intending to
drive all night, starting was postponed until near sundown. Two or
three miles from the Arkansas we apparently reached the general
altitude of the plains over which we trudged during the whole night,
with nothing but the rumbling of the wagons and the occasional
shout of one of the drivers to break the silence of the plain.
DIFFICULT TO GET THE HEAVILY LOADED WAGONS ACROSS.
It was my first experience of traveling at night, on this journey.

Toward midnight I became so sleepy that I could hardly sit on my
horse, so dismounting, I walked and led him. Advancing to a point
near the head of the trains I ventured to lie down on the ground to
rest, as the trains were passing at least. Instantly my clothes were
perforated with cactus needles which pricked me severely, and
waking me thoroughly. In the darkness it was with great difficulty I
could get the needles out. Mounting my horse again I rode some
distance in advance of everybody, completely out of hearing of the
trains, and riding thus alone, with nothing visible but the stars, a
feeling of melancholy seized me, together with a sense of
homesickness, with which I had not hitherto been troubled. Each
day’s travel was increasing the distance between me, my home and
my mother, to whom I was most dearly attached; and here amid the
solitude, darkness and perfect quietude of the vast plains I began to
reflect upon the dangers besetting me, and the uncertainty of ever
returning to my home or seeing my relatives again.
The approach of morning and the rising of the sun soon
dispelled these forebodings of evil and revived my spirits. Old Sol,
like a ball of fire, emerged from the endless plain to the east of us, as
from the ocean, soon overwhelming us with a flood of light such as I
had never experienced before. During all that day’s march the heat
was intense and the sunlight almost blinding, the kind of weather that
creates the mirage of the plains. In the distance on either hand, fine
lakes of clear water were seen glistening in the sun, sometimes
appearing circular in shape, surrounded with the proper shores, the
illusion being apparently complete, so much so that several times
during the day I rode some distance seeking to ascertain if they were
really lakes or not. I found them receding as I approached, and was
unable to get any closer to them than when as a boy I set out to find
the sack of gold at the end of the rainbow.
About midday we passed a great pile of bleached bones of
mules that had been thrown up in a conical shaped heap by the
passing trainmen, in the course of the ten years they had been lying
there. They were the remains of 200 or 300 mules belonging to John
S. Jones, a Missourian, a citizen of Pettis county, whom I knew
personally. In 1847, and for many years afterward, Jones was
engaged in freighting across the plains. In ’47, having obtained a
contract from the government to transport freight for the troops at
Santa Fé, he got a start late in the season, and had only reached the
crossing of the Arkansas when he was overtaken by such deep
snow and severely cold weather as to compel him to stop and go
into quasi-winter quarters. While there, protected by such barracks
for man and beast as could be hastily constructed, he received
orders from the commander of the troops in New Mexico that he
must hurry up with the supplies, orders of such urgency that they
could not be disregarded. He had a mule train of thirty wagons.
Orders were given to hitch up and start. The weather moderated the
first day, but on the second they encountered a heavy and cold rain
freezing as it fell, and were forced to go into corral. Intense cold
followed and every one of the mules froze to death, huddling in the
corral, during the night. Years afterwards, through the influence of
Colonel Benton in the Senate and John G. Miller of Missouri in the
House of Representatives an appropriation was made by Congress
of $40,000 to pay Mr. Jones for the loss of his mules.
In the forenoon of the second day from the Arkansas we reached
Sand creek, a tributary of the Cimarron, where we found a pool of
stagnant water, not enough for the oxen, but sufficient for the
trainmen to make coffee with, and there we camped. A few hours
afterwards we struck the valley of the Cimarron, and, after riding up
the bed of the apparently dry stream, we discovered a pool of clear
water. The cattle were so famished that they ran into it, hitched to the
wagons, their drivers being unable to restrain them, and it was with
considerable difficulty that the wagons were afterwards pulled out of
the mud.
VIII.
My First Antelope.
After reaching the Cimarron we began seeing herds of antelope

in the distance. At first I tried “flagging” them. I had been told that on
approaching within two or three hundred yards of them, concealed
from their view behind an intervening ridge, these animals were
possessed of such inordinate curiosity that they could be enticed to
within gunshot of the hunter by tying a handkerchief on the end of a
stick and elevating it in sight of the antelope, the hunter, of course,
keeping concealed. I made several efforts at this plan of exciting
their curiosity, and while some of them came toward me at first sight
of the flag, their curiosity seemed counterbalanced by caution or
incredulity, and in no instance could I get one to come near enough
for a sure or safe shot. I then tried a rifle, with which I was also
unsuccessful, not then being able to make a correct estimate of the
distance between me and the antelope, a troublesome task, only to
be acquired by experience and constant practice.

Advances in Protein Chemistry and Structural Biology Volume 115 Dna Repair Rossen Donev Full Chapter

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Advances in Protein Chemistry and Structural Biology Volume 115 Dna Repair Rossen Donev Full Chapter

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Advances in Protein Chemistry and

Structural Biology Volume 115 - DNA

First edition 2019

Copyright © 2019 Elsevier Inc. All rights reserved.

For information on all Academic Press publications

Publisher: Zoe Kruze

Marcel A.T.M. van Vugt

DNA repair by photolyases

oxidative damage, ultraviolet and other types of radiation, and hydrolytic

2. Cryptochrome/photolyase protein family

Fig. 2 Phylogenetic tree of the cryptochrome/photolyase family. The unrooted phylo-

identification of photoreactivation in Escherichia coli. Two genetic loci (phrA

2.1 Cofactors of the photolyases

Fig. 3 Structure of chromophores of photolyases. (A) Flavin adenine dinucleotide (FAD),

mononucleotide (FMN) as a second chromophore (Fig. 3A) (Ueda, Kato,

of FAD can be differentiated by absorption spectrum. Purified EcPL exhibits

2.2 The crystal structure of the photolyase

Fig. 5 Superimposition of CPD photolyases of Escherichia coli, Thermus thermophilus,

2.3 Reaction mechanism of DNA repair mediated by CPD

Fig. 7 Reaction mechanism of E. coli CPD photolyase. First, methenyltetrahydrofolate

To understand how this is achieved Tan et al. performed femtosecond spec-

2.4 Reaction mechanisms of DNA repair mediated by (6-4)

discovered in some other eukaryotic organisms. Therefore it is thought

Menck, C. F. (2002). Shining a light on photolyases. Nature Genetics, 32(3), 338–339.

TFIIH: A multi-subunit complex

In eukaryotes, NER requires the concerted action of more than 20 fac-

Fig. 1 See legend on opposite page.

Before reaching Pawnee Rock we overtook a train of thirty

Escaping any further delay from Indians or from other causes,

DIFFICULT TO GET THE HEAVILY LOADED WAGONS ACROSS.

It was my first experience of traveling at night, on this journey.

After reaching the Cimarron we began seeing herds of antelope

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