ADDIS ABABA UNIVERSITY

COLLEGE OF HEALTH SCIENCE

DEPARTMENT OF MEDICAL LABORATORY SCIENCE

Microbial genetics and molecular technique Individual assignment II

Module Code: CLAS 621

TITLE: DNA replication other than E.coli bacteria and regulation of DNA replication in
E.coli

Prepared by: Habtamu Molla

Id No: GSR/ 1888/15

Level----MSC , program---regular, year----I

Summitted to: Dr. Zewudu T.( PhD)

Submission Date:28/12/2022

Addis Ababa , Ethiopia, December 2022 G.C.

Acknowledgment

Firstly, I would like to say thanks to Almighty GOD and his mother for giving me all things.
Secondly, I would like to express deep gratitude to my respective teacher Dr. Zewudu
Terefework (PhD.), who give me the chance to do this assignment and for help by addressing
clear information on the way and methods we used to do this assignment. And also, I would like
to express my heartfelt thanks to all my group members who helps me by showing different
methods and also gives comment on my assignment.
Table of contents

Acknowledgment.........................................................................................................................................2

Table of contents.........................................................................................................................................3

Abbreviation and acronyms.........................................................................................................................4

List of figures...............................................................................................................................................5

1. DNA replication other than E.coli bacteria and regulation of DNA replication in E.coli...........................6

1.1.Introduction...................................................................................................................................6

1.2. DNA replication in Bacteria species other than E. coli (B.subtilis).................................................6

1.2.1. Initiation of DNA Replication......................................................................................................6

1.2.2. The Elongation Phase.................................................................................................................8

1.2.3.Termination of DNA Replication..........................................................................................9

1.3. Similarity and difference between DNA replication in E. coli and other bacteria........................10

1.3.1. Difference between E. coli and B. subtilis.........................................................................10

1.3.2.Similarity between E. coli and B. subtilis............................................................................10

1.4. Regulation of DNA replication in E. coli.......................................................................................11

1.4.1.Regulatory Inactivation of DnaA (RIDA).............................................................................11

1.4.2. DnaA-Box Sequences: datA and DARS..............................................................................11

1.4.3. IHF (DNA-bending proteins integration host factor) and Fis.............................................12

1.4.4. Regulation of DNA replication by DNA methylation (SeqA)..............................................12

1.4.5. Lysine Acetylation of DnaA...............................................................................................13

Summary...................................................................................................................................................14

Conclusion.................................................................................................................................................15

References.................................................................................................................................................16
Abbreviation and acronyms

ADP- adenosine triphosphate

ATP- adenosine diphosphate

B.Subtilis- Bacteria named Vibrio subtilis

DARS1- DnaA-reactivating site 1

DARS2- DnaA-reactivating site 2

DNA- Deoxyribose nucleic acid
OriC- origin of replication

Pol C- Polymerase C

Pol III- polymerase III

SSB- Single strand binding protein

 List of figures

Figure 1 -Initiation of DNA replication in B. subtilis in contrast with E. coli..................................................7

Figure 2 -DNA replication: Elongation phase of two organisms, E. coli Vs B. subtilis..................................8

Figure 3 -Termination of DNA replication in B. subtilis................................................................................9

Figure 4 - DNA replication regulatory inactivation of DnaA (RIDA) in E. coli.................................................11

Figure 5 - Regulation of DNA replication by DNA Adenine methylation (SeqA) in E. coli..............................13

List of tables
Table 1 - Difference in protein used by E. coli and B. subtilis in case of DNA replication...........................10
1. DNA replication other than E.coli bacteria and regulation of DNA

replication in E.coli

1.1.Introduction

The replication origin and the initiator protein DnaA are the main targets for regulation of
chromosome replication in bacteria. The origin bears multiple DnaA binding sites, while DnaA
contains ATP/ADP-binding and DNA-binding domains. When enough ATP-DnaA has
accumulated in the cell, an active initiation complex can be formed at the origin resulting in
strand opening and recruitment of the replicative helicase .(1) DNA replication in Bacteria
possesses three distinct phases: - initiation, elongation and termination. In bacteria, DNA
replication is initiated by the binding of a protein initiator, DnaA, to the origin of replication,
oriC. During the elongation phase of DNA replication, DNA is synthesized by the action of a
large multi-subunit complex known as the replisome. The termination of DNA replication occurs
at a termination locus positioned directly opposite oriC .(5,12,15) There are a number of
similarities between B. subtilis and E. coli. for example, similar protein structure used by both of
them (primase both uses DnaG) and differ in Helicase protein (E. coli uses DnaB, whereas B.
subtilis uses DnaC). beside this there are a lot of difference and similarity between them. The
regulation of DNA controlled by a series of mechanisms, like Regulatory Inactivation of DnaA
(RIDA), Regulation of DNA replication by DNA methylation (SeqA). (19,36)
1.2. DNA replication in Bacteria species other than E. coli (B.subtilis)
The process of DNA replication can be separated into three distinct phases:
 Initiation
 Elongation
 Termination.
During the initiation phase, a nucleoprotein complex assembles at the origin of replication. In the
elongation phase, carried out template-directed DNA synthesis. This is continuous and
processive on the leading strand, but discontinuous on the lagging strand where a more complex
cycle of primer synthesis, strand elongation and fragment ligation take place. Finally, during
termination, DNA polymerization is paused at a specific termination site. Regulation of DNA
replication occurs principally at the initiation stage.
1.2.1. Initiation of DNA Replication

In bacteria, DNA replication is initiated by the binding of a protein initiator, DnaA, to the origin
of replication, oriC. DnaA is a right-handed helical oligomer on the DNA (2,3) directed by its
binding to a series of recognition sites within the origin termed DnaA-boxes (4). The formationof
this oligomer brings unwinding of the DNA duplex within the origin at an AT-rich region ,called
DUE (DNA Unwinding Element) (5,6). DnaA then recruiting DNA helicase ( DnaB in E. coli or
DnaC in B. subtilis), which is loaded onto the unwound single-stranded DNA (ssDNA) by
ahelicase loader, DnaC for E. coli and DnaI for B. subtilis (7).
Figure 1-Initiation of DNA replication in B. subtilis in contrast with E. coli
In B. subtilis, initiation requires two additional essential proteins, DnaD and DnaB, both ofwhich
possess DNA remodelling activities and bind to the origin prior to helicase loading (18). DnaD is
used for double-stranded DNA (dsDNA) melting, while DnaB have a role in helicase loading
(8,9).

1.2.2. The Elongation Phase

During the elongation phase of DNA replication, DNA is synthesized by the action of a large
multi-subunit complex known as the replisome. Elongation in B. subtilis occurs by a similar
mechanism; however, it uses two different, but related DNA polymerases, PolC and DnaE. DnaE
is more related to E. coli Pol III.These polymerases essential for lagging strand synthesis, while
PolC is required for leading strand synthesis (15).
Each DNA polymerase can extend DNA primers, but DnaE extend the RNA primers produced
by DNA primase. DnaE extends the RNA primers with DNA before passing to PolC for further
strand synthesis (10,11).

Figure 2-DNA replication: Elongation phase of two organisms, E. coli Vs B. subtilis

1.2.3.Termination of DNA Replication

The termination of DNA replication occurs at a termination locus positioned directly opposite
oriC. In B. subtilis, the binding of two homodimers replication termination protein (RTP) at ‘A’
and ‘B’ sites within the termination region is required to arrest replication (12). The approach of
the replication machinery from the ‘B’ site results in termination of replication (non-permissive
direction) while from the ‘A’ site allows replication to continue (permissive direction). A-site
binding is also essential to block replication fork progression, and A-site binding by RTP is
following by B-site binding (13).

Figure 3 -Termination of DNA replication in B. subtilis

1.3. Similarity and difference between DNA replication in E. coli and other
bacteria

1.3.1. Difference between E. coli and B. subtilis

Table 1- Difference in protein used by E. coli and B. subtilis in case of DNA replication

The cellular DnaA–ATP concentration plays an important role in the regulation of E. coli
replication initiation. Many of the regulatory mechanisms in E. coli found to influence the
adenosine nucleotide bound state of DnaA (14). B. subtilis, by contrast, no regulator had
identified which affects the conversion of DnaA–ATP to DnaA–ADP. Instead, replication
regulators in B. subtilis act directly on Genes. This evidence suggests that the primary
mechanisms of DNA replication control are different in B. subtilis and E. coli (15). The initiation
regulators determine the ligation status (ATP versus ADP) of DnaA in E. coli while in B. subtilis
they act to control DnaA oligomerisation at oriC.

1.3.2.Similarity between E. coli and B. subtilis

 Both organisms utilize a major regulator during vegetative growth which
interacts with both DnaA and DnaN; YabA in B. subtilis and Hda in E. coli.
 Both organisms have regulators which are concerned in chromosome segregation,
SeqA in E. coli, and Soj and SirA during growth and sporulation of B. subtilis,
respectively.
 Both organisms utilize a method of origin sequestration to prevent DnaA
binding.
 Furthermore, both organisms have advanced a regulator which targets a
structurally equivalent location on DnaA domain I.
 Thus, these may represent common feature of replication regulation between
E. coli and B. subtiles (16,17).
1.4. Regulation of DNA replication in E. coli
DNA replication is controlled by a series of mechanisms that are adjusted on the control of
cellular DnaA levels.

1.4.1.Regulatory Inactivation of DnaA (RIDA)

One of the main mechanisms called RIDA system (Regulatory Inactivation of DnaA). This
mechanism takes place when DnaA is activated by its binding to ATP. The accumulation of
DnaA in this active form leads to the initiation of replication since it facilitates its binding to the
oriC on the DNA. DnaA changes to its inactive form DnaA-ADP by hydrolysis of ATP.
HdaADP is responsible for promoting the hydrolysis of DNA-ATP (19). This inactivating
regulation of DnaA is key for preventing the over-initiation of replication during the cell cycle.

Figure 4- DNA replication regulatory inactivation of DnaA (RIDA) in E. coli

1.4.2. DnaA-Box Sequences: datA and DARS

Another mechanism, that regulates the initiation of DNA replication is by controlling the
availability of free DnaA to bind to DnaA boxes on the oriC. E. coli possesses three loci with
DnaA-box motifs that are used to regulate DNA replication initiation. datA, ≈1 kb in length,
contains five DnaA-boxes with high affinity for DnaA. It acts as a regulatory of DNA replication
initiation. Deletion of datA or mutations of the DnaA-boxes within datA causes over- initiation
of DNA replication (20-23). Binding of IHF to datA promotes DnaA- binding and is essential for
the regulatory action. A recent study showes that datA promotes the hydrolysis of DnaA–ATP to
DnaA– ADP (24). datA locus localized near from the oriC, shows high affinity for DnaA, even
more than the DnaA boxes on the oriC. Thus, the datA region is able to bind over 300 DnaA
molecules whereas oriC binds to 45 DnaA monomers (25-27). At high levels DnaA binds to the
DnaA boxes in the promoter region and impedes transcription. This auto-repressive process
directly affects the amount of DnaA- ATP available and controls the initiation of DNA
replication (28). Two other loci in E. coli, termed DARS1 (DnaA-reactivating site 1) and
DARS2 (DnaA-reactivating site 2). Those loci used for reactivation of DnaA by promoting
nucleotide exchange, generating DnaA– ATP from DnaA–ADP (5). Deletion of DARS
sequences causes inhibition of DNA replication due to a decrease DnaA–ATP concentration
(29).

1.4.3. IHF (DNA-bending proteins integration host factor) and Fis

The DNA-bending proteins integration host factor (IHF) and Fis are play important roles in
regulating the binding of DnaA at the origin of replication (30). Fis has used as both inhibitory
and stimulatory roles in DNA replication initiation (31,32). Both proteins bind to oriC and act in
an antagonistic manner (30,33).
Binding of IHF to a replication origin, promotes binding of DnaA at DnaA-boxes within
theorigin, contributing to DnaA oligomer formation. The binding of IHF encourages a bend in
the DNA, which bring the two adjacent DnaA-boxes into closer, facilitating the extension of the
helical DnaA oligomer (34). Fis also inhibit DNA unwinding at oriC by blocking binding of both
DnaA and IHF. Increasing concentrations of DnaA were found to relieve Fis inhibition and IHF
redistribute DnaA molecules at oriC (30).

1.4.4. Regulation of DNA replication by DNA methylation (SeqA)

For initiation of DNA replication, both DNA strands should be methylated, this process is
mediated by Dam (DNA adenine methyl tranferase) (35). Dam binds to the DNA non
specifically, and methylates the GATC sites. The methylation process occurs on the newly
synthesized strands and on the lagging, strand occurs only after the ligation of the Okazaki
fragments. Dam is always located near to the replication origin, so that the methylation of DNA
strands occurs as soon as polymerization begins. The presence of hemi methylated GATC sites
indicate that DNAreplication has just occurred (36). SeqA represses the replication of DNA by
binding to the hemi methylated GATC sequence at the oriC, due to overlapping of SeqA DNA-
binding siteswith DnaA (DnaA boxes) on the oriC, impedes the complete access of DnaA-ATP
to the oriC. This prevention of replication (37). Dam binds to the DNA non specifically, and
methylates the GATC sites. The methylation process occurs on the newly synthesized strands
and on the lagging, strand occurs only after the ligation of the Okazaki fragments. Dam is always
located near to the replication origin, so that the methylation of DNA strands occurs as soon as
polymerization begins. The presence of hemi methylated GATC sites indicate that DNA
replication has just occurred (36). SeqA represses the replication of DNA by binding to the hemi
methylated GATC sequence at the oriC, due to overlapping of SeqA DNA-binding sites with
DnaA (DnaA boxes) on the oriC, impedes the complete access of DnaA-ATP to the oriC. This
prevention of replication (37).
Figure 5- Regulation of DNA replication by DNA Adenine methylation (SeqA) in E. coli

1.4.5. Lysine Acetylation of DnaA

A recently discovered mechanism for controlling DNA replication initiation in E. coli involves
acetylation of lysines within DnaA. Acetylation sites were identified on 13 lysines expressed
DnaA, including: lysine (Lys178) required for the binding of ATP (37). Mutation of Lys178 to
Gln or Arg prevented ATP-binding to DnaA, similarly acetylation of Lys178 have a similar
effect in inactivating the initiator. Generally, the cellular DnaA–ATP concentration plays an
important role in the regulation of E. coli replication initiation (6). Many of the regulatory
mechanisms in E. coli found to influence the adenosine nucleotide bound state of DnaA (13).
The datA, DARS1 and DARS2 loci influence available DnaA–ATP levels by promoting ATP-
hydrolysis (datA) or nucleotide exchange from ADP to ATP (DARS) (29). lysine acetylation of
DnaA affect ATP-binding to DnaA and thus inhibit initiation of DNA replication. (36)
Summary

 DNA replication in Bacteria possesses three distinct phases: - initiation, elongation and
termination.
 Initiated of DNA replication trigger by the binding of a protein initiator, DnaA, to the origin
of replication, oriC.
 During the elongation phase of DNA replication, DNA is synthesized by the action of a
large multi-subunit complex known as the replisome.
 The termination of DNA replication occurs at a termination locus positioned directly
opposite oriC.
 There are a number of similarities between B. subtilis and E. coli.
 The regulation of DNA controlled by a series of mechanisms, like Regulatory Inactivation of
DnaA (RIDA), Regulation of DNA replication by DNA methylation (SeqA) etc.
Conclusion
Generally, Multiple systems regulate oriC and DnaA in a concerted manner to ensure that
replication initiation occurs only once per origin per generation. Some of these regulatory
systems are coupled with specific events that are important for cell cycle regulation or
chromosomal replication.there are three distinct phases in an organism’s DNA replication;
initiation, elongation and termination. Initiation: helicase unwinds double helix DNA in to
RNA.
Elongation; new DNA stands grows base by base starting at primes Termination; last primer
sequence is removed from the end of lagging strand.in order to stop DNA replication. in the
regulation of E. coli replication initiation, the cellular DnaA–ATP concentration plays an
important role. Many of the regulatory mechanisms in E. coli found to influence the adenosine
nucleotide bound state of DnaA. Hda, along with the DNA polymerase clamp, DnaN, acts to
promote the hydrolysis of DnaA– ATP after initiation.
References

1. Skarstad, K., & Katayama, T. (2013). Regulating DNA replication in bacteria. Cold

Spring Harbor perspectives in biology, 5(4), a012922.

2. Zorman, S.; Seitz, H.; Sclavi, B.; Strick, R. Topological characterization of the DnaA-

oriC complex using single-molecule nanomanipuation. Nucleic Acids Res. 2012, 40,

7375–7383.

3. Erzberger, J.P.; Mott, M.L.; Berger, J.M. Structural basis for ATP-dependent DnaA

assembly and replication-origin remodeling. Nat. Struct. Mol. Biol. 2006, 13, 676–683.

4. Fuller, R.S.; Funnell, B.E.; Kornberg, A. The dnaA protein complex with the

E. coli chromosomal replication origin (oriC) and other DNA sites. Cell 1984, 38, 889–

900.

5. Kowalski, D.; Eddy, M.J. The DNA unwinding element: A novel, cis-acting component

that facilitates opening of the Escherichia coli replication origin. EMBO J. 1989, 8,

4335–4344.

6. Bramhill, D.; Kornberg, A. Duplex opening by dnaA protein at novel sequences in

initiation of replication at the origin of the E. coli chromosome. Cell 1988, 52, 743–755

7. Marszalek, J.; Kaguni, J.M. DnaA protein directs the binding of DnaB protein in

initiation of DNA replication in Escherichia coli. J. Biol. Chem. 1994, 269, 4883–

4890.

8. Bruand, C.; Ehrlich, S.D.; Jannière, L. Primosome assembly site in Bacillus subtilis.

EMBO J. 1995, 14, 2642–2650.

9. Zhang, W.; Carneiro, Roberts, C.J.; Soultanas, P. The Bacillus subtilis DnaD and DnaB

proteins exhibit different DNA remodelling activities. J. Mol. Biol. 2005, 351, 66–75.
10.Dervyn, E.; Suski, C.; Daniel, R.; Bruand et al. Two essential DNA polymerases at the

bacterial replication fork. Science 2001, 294, 1716–1719.

11.Sanders, G.M.; Dallmann, H.G.; McHenry, C.S. Reconstitution of the B. subtilis

Replisome with 13 Proteins Including Two Distinct Replicases. Mol. Cell 2010, 37, 273–

2 12. Hill, T.M. Arrest of bacterial DNA replication. Annu. Rev. Microbiol. 1992, 46, 603–

633.

13. Langley, D.B.; Smith, M.T.; Lewis, P.J.; Wake, R.G. Protein-nucleoside contacts in

the interaction between the replication terminator protein of Bacillus subtilis and the

DNA terminator. Mol. Microbiol. 1993, 10, 771– 779.

14. Kato, J.; Katayama, T. Had a novel DnaA-related protein, regulates the replication

cycle in Escherichia coli. EMBO J. 2001, 20, 4253–4262.

15. Okumura, H.; Yoshimura, M.; Ueki, M.; Oshima, T.; Ogasawara, N et al. Regulation

of chromosomal replication initiation by oriC-proximal DnaA- box clusters in Bacillus

subtilis. Nucleic Acids Res. 2012, 40, 220–234.

16. Noirot-Gros, M.-F, N, Polard, P.; Noirot, P. Functional dissection of YabA, a negative

regulator of DNA replication initiation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA

2006, 103, 2368–2373

17. Kurz, M.; Dalrymple, B.; Wijffels, G.; Kongsuwan, K. Interaction of the Sliding Clamp

Beta-Subunit and Hda, a DnaA-Related Protein. J. Bacteriol. 2004, 186, 3508–3515

18. 30.Leonard, A. C. & Grimwade, J. E. Initiating chromosome replication in

E. coli: It makes sense to recycle. Genes Dev. 2009, 23, 1145-115

19. Katayama, T., Kubota, T., Kurokawa, T., Crooke, E. & Sekimizu, K. The initiator

function of DnaA protein is negatively regulated by the sliding clamp of the E. coli

chromosomal replicase. Cell. 1998, 94, 61-71.

20. Kitagawa, R.; Mitsuki, H.; Okazaki, T.; Ogawa, T. A novel DnaA protein- binding

site at 94.7 min on the Escherichia coli chromosome. Mol. Microbiol. 1996, 19, 1137–

1 21.Kitagawa, R.; Ozaki, T.; Moriya, S.; Ogawa, T. Negative control of replication

initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia

coli DnaA protein. Genes Dev. 1998, 12, 3032– 3043.

22. Ogawa, T.; Yamada, Y.; Kuroda, T.; Kishi, T.; Moriya, S. The datA locus

predominantly contributes to the initiator titration mechanism in the control of

replication initiation in Escherichia coli. Mol. Microbiol. 2002, 44, 1367– 137

1523. Kasho, K.; Katayama, T. DnaA binding locus datA promotes DnaA-ATP hydrolysis to

enable cell cycle-coordinated replication initiation. Proc. Natl. Acad. Sci. USA 2012,

110, 936–941.

24. Kitagawa, R., Ozaki, T., Moriya, S. & Ogawa, T. Negative control of replication

initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia

coli DnaA protein. Genes Dev. 1998, 12, 3032-3043.

25.Messer, W. & Weigel, C. DnaA initiator Ɇ also a transcription factor. Mol. Microbiol.

1997, 24, 1-6

26.Gama-Castro, S., Salgado, H., Peralta-Gil, M et al. RegulonDB version 7.0:

transcriptional regulation of Escherichia coli K-12 integrated within genetic sensory

response units (Gensor Units). Nucleic Acids Res. 2010, 39, D98- D105.

27.Mott, M. L. & Berger, J. M. DNA replication initiation: mechanisms and regulation

in bacteria. Nature Rev Microbiol. 2007, 5, 343-54.

28.Fujimitsu, K.; Senriuchi, T.; Katayama, T. Specific genomic sequences of E. coli

promote replicational initiation by directly reactivating ADP-DnaA. Genes Dev. 2009,

23, 1221–1233.

29.Ryan, V.T.; Grimwade, J.E.; Camara, J.E.; Crooke, E.; Leonard, A.C. Escherichia coli

prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and

DnaA. Mol. Microbiol. 2004, 51, 1347–1359.

30.Filutowicz, M.; Ross, W.; Wild, J.; Gourse, R.L. Involvement of Fis protein in

replication of the Escherichia coli chromosome. J. Bacteriol. 1992, 174, 398–407.

31.Pratt, T.S.; Steiner, T.; Feldman, L.S.; Walker, K.A.; Osuna, R. Deletion analysis of

the fis promoter region in Escherichia coli: Antagonistic effects of integration host

factor and Fis. J. Bacteriol. 1997, 179, 6367–6377

32.Cassler, M.R.; Grimwade, J.E.; Leonard, A.C. Cell cycle-specific changes in

nucleoprotein complexes at a chromosomal replication origin. EMBO J. 1995, 14, 5833–

58 33.Kaur, G.; Vora, M.P.; Czerwonka, C.A.; Rozgaja, T.A.; Grimwade, J.E.; Leonard,

A.C. Building the bacterial orisome: High affinity DnaA recognition plays a role in

setting the conformation of oriC DNA. Mol. Microbiol. 2014, 91, 1148–1163.

34.Wion, D. & Casadésus, J. N6-methyl-adenine: an epigenetic signal for

DNAoprotein interactions. Nature Rev Microbiol. 2006, 4, 183-192.

35.Lu, M., Campbell, J. L., Boye, E. & Kleckner, N. SeqA: a negative modulator

of replication initiation in E. coli. 1994, Cell 77, 413-426 41147. 81.

36.Han, J. S., Kang, S., Kim, S. H., Ko, M. J. & Hwang, S. Binding of SeqA protein to

hemi-methylated GATC sequences enhances their interaction and aggregation properties.

J. Biol. 2004, Chem 279, 30236-30243.

37.Zhang, Q.; Zhou, A.; Li, S.S.; Ni, J et al. Reversible lysine acetylation is involved in

DNA replication initiation by regulating activities of initiator DnaA in Escherichia

coli. Sci. Rep. 2016,6,3083-30