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J.Mol.Biol. 2007, 366, 1387-1400

J.Mol.Biol. 2007, 366, 1387-1400

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10/06/2009

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This article was originally published in a journal published byElsevier, and the attached copy is provided by Elsevier for theauthor’s benefit and for the benefit of the author’s institution, fornon-commercial research and educational use including withoutlimitation use in instruction at your institution, sending it to specificcolleagues that you know, and providing a copy to your institution’sadministrator.All other uses, reproduction and distribution, including withoutlimitation commercial reprints, selling or licensing copies or access,or posting on open internet sites, your personal or institution’swebsite or repository, are prohibited. For exceptions, permissionmay be sought for such use through Elsevier’s permissions site at:http://www.elsevier.com/locate/permissionusematerial
 
   A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y
Examination of the Long-range Effects ofAminofluorene-induced Conformational Heterogeneityand Its Relevance to the Mechanism of TranslesionalDNA Synthesis
Srinivasarao Meneni, Fengting Liang and Bongsup P. Cho
Department of Biomedical andPharmaceutical Sciences,College of Pharmacy, Universityof Rhode Island, Kingston,RI 02881, USA
Adduct-induced conformationalheterogeneity complicatesthe understand-ing of how DNA adducts exert mutation. A case in point is the
-deacetylated AF lesion [
-(2
-deoxyguanosin-8-yl)-2-aminofluorene], themajor adduct derived from the strong liver carcinogen
-acetyl-2-aminofluorene. Three conformational families have been previouslycharacterized and are dependent on the positioning of the aminofluorenerings: B is in the
B
-DNA
major groove, S is
stacked
into the helix with base-displacement, and W is
wedged
into the minor groove. Here, weconducted
19
F NMR, CD,
m
, and modeling experiments at various primerpositions with respect to a template modified by a fluorine tagged AF-adduct(FAF).Inthefirstset,theFAF-GwaspairedwithCandinthesecondset it was paired with A. The FAF-G:C oligonucleotides were found topreferentially adopt the B or S-conformers while the FAF-G:A mismatchones preferred the B and W-conformers. The conformational preferences of  both series were dependent on temperature and complementary strandlength; the largest differences in conformation were displayed at lowertemperatures. The CD and
m
results are in general agreement with theNMR data. Molecular modeling indicated that the aminofluorene moiety inthe minor groove of the W-conformer would impose a steric clash with thetight-packing amino acid residues on the DNA binding area of the
Bacillus
fragment (BF), a replicative DNA polymerase. In the case of the B-typeconformer, the carcinogenic moiety resides in the solvent-exposed majorgroove throughout the replication/translocation process. The presentdynamic NMR results, combined with previous primer extension kineticdata by Miller & Grollman, support a model in which adduct-inducedconformational heterogeneities at positions remote from the replication forkaffect polymerase function through a long-range DNA
protein interaction.
© 2006 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords:
aminofluorene-DNA adducts; conformational heterogeneity;long-range effect; tranlesion synthesis
Introduction
DNA adduct formation is a signature hallmarkof mutation, ultimately leading to the initiation of chemical carcinogenesis.
1,2
Arylamines and theirnitro derivatives are a major group of mutagensand carcinogens.
3,4
The environmental carcinogen2-nitrofluorene and its amino derivatives are theprototype arylamine carcinogens.
5,6
Upon activa-tion
in vivo
, they react with cellular DNA to formtwo major C8-substituted dG adducts:
-(2
-deoxyguanosin-8-yl)-2-acetyaminofluorene (AAF)
1
Abbreviations used: AF-adduct,
-(2
-deoxyguanosin-8-yl)-2-aminofluorene;AAF-adduct,
-(2
-deoxyguanosin-8-yl)-2-acetylaminofluorene; BF,
Bacillus
fragment; CD, circulardichroism; FAF, 7-fluoro-2-aminofluorene; ICD
290-360 nm
,induced circular dichroism at 290
360 nm; SMI, slippedmutagenic intermediates; TLS, translesion synthesis;NOESY, nuclear Overhauser effect spectroscopy; H/D,hydrogen/deuterium.E-mail address of the corresponding author: bcho@uri.edu
doi:10.1016/j.jmb.2006.12.023
J. Mol. Biol.
(2007)
366
, 1387
1400
0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
 
   A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y
and
-(2
-deoxyguanosin-8-yl)-2-aminofluorene(AF) (Figure 1(a)), along with the minor 3-(2
-deoxyguanosin-
2
-yl)-2-acetylaminofluorene (dG-
2
-AAF).
7
These bulky DNA lesions, if notrepaired, can produce mutations during replicationthat initiate the process of carcinogenesis. In bacteria AF-adducts mostly induce G
T transver-sions, whereas AAF-adducts cause mostly deletionmutations.
3
In mammalian cells, however, bothadducts promote sequence-dependent base substi-tution mutations.
8
The bulky AF and AAF adducts have long beenused as model systems for investigation of thestructure/functionion relationship in the arylaminemutagen family.
1
4
However, the molecular under-standing of how they exert mutation
in vivo
isgenerally poor and is complicated by adduct-induced conformational heterogeneities. The AAF-adduct exists predominantly in the base-displaced
stacked
(S) conformation.
9
11
Because it lacksWatson
Crick base-pairs at the lesion site, theAAF-adduct produces a major structural distortionin DNA. In contrast, the
-deacetylated AF-adductpossesses flexiblility around the glycosidyl bond(
χ
), enabling it to adopt multiple conformationalmotifs depending upon the location of the amino-fulorene moiety, including a major-groove binding
B-type
(B) conformation and a minor-groove binding
wedged
(W) conformation in additionto the S conformation (Figure 1(b)).
12
18
In general,fully base-paired DNA duplexes that have incorpo-rated AF are present in an S/B equilibrium in whichthe tendencyforthe molecules tofavorthe Sor theBconformation is dependent upon the flanking-sequence context.
17
19
Consistent with this observa-tion, the mutational specificity and frequency of theAF-adduct in mammalian cells vary dependingupon thesequence context inwhich itisembedded.
8
The W-conformer has only been observed in AF-modified duplexes with dA and dG-mismatches atthe lesion site;
20
22
these mispairings apparentlyunderlieG
Tand G
Ctransversions, respectively.Normally, DNA replication in a replicative poly-merase proceeds with high fidelity and processivityon a natural DNA template.
23
26
The process, how-ever, is interupted significantly when a damaged base is present in the template. Continued replica-tion of the adduct-containing template is termed astranslesion synthesis (TLS), which is a major sourceof point mutations.
27,28
The TLS events are modu-latedbyvariousfactorsincludingthelesion-inducedconformational changes of the template, its sur-rounding base sequence context, and by the natureof DNA polymerases.
28,29
The slowing of replicationin a replicative polymerase is now understood topredominantly produce switch to a lesion bypasspolymerase
in vivo,
which is frequently error prone.However, many specific details of this paradigmremains unknown, i.e. how does the polymeraseswitch at a site of DNA damage really occur?
27
Mutagenic outcomes in replicative polymerasesmay alsocontribute tothe overall mutagenic burden
in vivo
.
30
Although the steady-state kinetics of nucleotideinsertion opposite the lesion have been studiedextensively for many DNA adducts,
31
34
data deal-ing with the long-range effects of a lesion on TLS arelimited.
35
39
Lindsley & Fuchs
36
have shown thatthe rate of primer extension by T7 DNA polymerase both at (n) and adjacent to (n+1) the lesion site witha DNA template containing an AF-lesion is reducedsignificantly (
10
4
) relative to unmodified controlDNA. A greater rate reduction (
10
6
) was ob-served in the presence of an AAF-adduct. Miller &Grollman
37
subsequently extended the scope of theinvestigation, probing the long-range effects of AF and other DNA adducts on the exo
Klenowfragment of 
Escherichia coli
DNA polymerase I.Polymerase activity was affected as far as four bases downstream (n
n+3)(10
4
10
6
) of the dG[AF]:dA mismatch lesion. The effect was much lesspronounced (10
1
10
2
) for extension of dG [AF]:dC. Similar results were obtained with Pol II & III.
39
The slowed replication rate even after incorporationof bases opposite the lesion provides sufficient timefor template alignment, a general model to predictdeletion mutations by various bulky DNA adductsincluding AF and AAF-adducts.
28,31
In the present study we have used the well-defined AF-induced B/S/W-conformational hetero-geneity model
17,40
as a basis for investigating theadduct structures at various positions relative to theprimer terminus during simulated AF-induced TLS.Temperature-dependent
19
F NMR spectra wereobtained for two distinct TLS models: extension of dG [FAF]:dC-match and dG [FAF]:dA mismatch.The dynamic NMR results coupled with the model-ing work with the thermophilic DNA polymerase I
Bacillus
fragment (BF)
41
were examined in thecontext of previous kinetic parameters
36,37
in orderto gain insight into the long-range effects of theconformationally flexible AF-adduct on DNA poly-merase function.
Results
The synthesis and purification of an FAF-mod-ified 12-mer oligodeoxynucleotide and time-of-flight-mass spectrometry characterization were car-ried out using the procedures described.
19
Themodified template strand was annealed withappropriate primers in order to produce variousTLS models of the primer extension reaction (n
1,n, n+1, n+3, and n+6, n=primer terminus) (Figure1(c)).Figures 2 and 3show the dynamic
19
F NMRresults for the dC-match and dA-mismatch exten-sion models, respectively. We have recently demon-strated the utility of 
19
F NMR/CD procedure forprobing the AF-induced B-S-W-conformationalheterogeneities.
40
The
19
F NMR method takesadvantage of the sensitivity of the fluorine nucleusto the tertiary structure of DNA, thereby requiringfluorine-tagged FAF as a model probe (Figure1(a)).
12
Induced circular dichroism in the 290
360 nm FAF-absorbing range (ICD
290
360 nm
) serves
1388
Long-range Effect of Aminofluorene-Heterogeneity 

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