DNA STRUCTURE AND DNA

REPLICATION IN PROKARYOTES

2024

SUBMISSION OF DISSERTATION PROJECT WORK FOR

THE DEGREE OF MASTER OF SCIENCE IN

ZOOLOGY

DR. AKHTAR HASAN RIZVI SHIA P.G. COLLEGE

SUPERVISOR SUBMITTED BY
DR. SYED ZAKIR HUSAIN JANHAVI VISHWAKARMA

HEAD OF DEPARTMENT OF ZOOLOGY M.Sc. 2 SEMESTER (ZOOLOGY)

DR. A.H.R. SHIA PG. COLLEGE, JAUNPUR E.NO.- PU20/123380

 DR. AKHTAR HASAN RIZVI (SHIA) P G COLLEGE,
JAUNPUR

Dr. Syed Zakir Husain

Head, Department of Zoology

Dr. AHR Shia PG college

Jaunpur-222001 Contact: Mob. 9335073630

CERTIFICATE

I have great pleasure in forwarding this dissertation project work of Janhavi Vishwakarma
entitled the study of "DNA STRUCTURE AND DNA REPLICATION IN
PROKARYOTES" during the study of M.Sc II semester in Zoology to
V.B.S.Purvanchal Univercity Jaunpur.

Janhavi Vishwakarma has put more than 6 months for the project work in the laboratory
under my guidance and in my knowledge and belief the dissertation project embodies the
work done by the candidates herself.

Forwarded

Dr. Syed Zakir Husain Rizvi

Head Department of Zoology

Dr.Akhtar Hasan Rizvi (Shia) P.G.College

Jaunpur (U.P)
 Acknowledgement

At the outset of welcome this opportunity to the great pleasure of

expressing my sincere and heartly gratitude to dissertation project Dr.
Sayyed Zakir Husain Head, Department of Zoology, A.H.R. Shia
Degree college, V.B.S.P.U. Jaunpur for his valuable guidance,
encouragements, set high standard of inspiration, solving critical
problems and & genericity that bestowed up the students throughout
the tenure of present work.

I sincerely thanks to Dr. Sanjay kr. Pandey, Dr. Anurag Mishra and
Miss Vishakha Jaiswal, Sr. Lecturer Department of Zoology for their
academic helps in the laboratory and for the valuable suggestions and
guidance during the work.

I would like to convey gratitude towards my father Mr. Munna Lal

Vishwakarma and my mother Mrs. Sushma Vishwakarma, who
inspired and promote me to achieve my target.

Janhavi Vishwakarma

(M.Sc.- 2nd semester, Zoology)

 PREFACE

In the preparation of this project of zoology on the topic

" DNA STRUCTURE AND DNA REPLICATION IN

PROKARYOTES ", I have precisely demarcated all the

important points. I have made my best possible efforts
to remove all the errors.

It is a great pleasure for me to thank all those valuable

suggestions that have been given to me by Dr. Syed
Zakir Husain Sir. I must thank the almighty for this
inspiration and guidance as well as my parents, teachers
who directed me to complete this project file.
 Contents

 Introduction
 Regulation
 Elongation
 Rate of Replication
 Termination
 Rolling Circle Replication
 Components
 DNA Replication
 DNA Structure
 Reference
 DNA STRUCTURE AND DNA
REPLICATION IN PROKARYOTES

INTRODUCTION

All cells must finish DNA replication before they can proceed for cell

division. Media conditions that support fast growth in bacteria also

couples with shorter inter-initiation time in them, i.e. the doubling

time in fast growing cells is less as compared to the slow growth. In

other words, it is possible that in fast growth conditions the

grandmother cells starts replicating its DNA for grand daughter cell.

For the same reason, the initiation of DNA replication is highly

regulated. Bacterial origins regulate orisome assembly, a nuclei-

protein complex assembled on the origin responsible for unwinding

the origin and loading all the replication machinery. In E. coli, the

direction for orisome assembly are built into a short stretch of

nucleotide sequence called as origin of replication (oriC) which

contains multiple binding sites for the initiator protein DnaA (a

highly homologous protein amongst bacterial kingdom).

Dna A has four domains with each domain responsible for a specific

task. There are 11 DnaA binding sites/boxes on the E. coli origin of

replication out of which three boxes R1, R2 and R4 (which have a

highly conserved 9 bp consensus sequence 5' TTATC/ACACA are

high affinity DnaA boxes. They bind to DnaA-ADP and DnaA-ATP

with equal affinities and are bound by DnaA throughout most of the

cell cycle and forms a scaffold on which rest of the orisome

assembles. The rest eight DnaA boxes are low affinity sites that

preferentially bind to DnaA- ATP. During initiation, DnaA bound to

high affinity DnaA box R4 donates additional DnaA to the adjacent

low affinity site and progressively fill all the low affinity DnaA

boxes. Filling of the sites changes origin conformation from its native

state. It is hypothesized that DNA stretching by DnaA bound to the

origin promotes strand separation which allows more DnaA to bind to

the unwound region. The DnaC helicase loader then interacts with the

DnaA bound to the single-stranded DNA to recruit the Dnaß helicase,

which will continue to unwind the DNA as

the DnaG primase lays down an RNA primer and DNA Polymerase

III holoenzyme begins elongation.

REGULATION

Chromosome replication in bacteria is regulated at the initiation stage.

DnaA-ATP is hydrolyzed into the inactive DnaA-ADP by RIDA

(Regulatory Inactivation of DnaA), and converted back to the active

DnaA- ATP form by DARS (DnaA Reactivating Sequence, which is

itself regulated by Fis and IHF). However, the main source of DnaA-

ATP is synthesis of new molecules. Meanwhile, several other proteins

interact directly with the oric sequence to regulate initiation, usually

by inhibition. In E. coli these proteins include DiaA, SeqA, IciA, HU,

and ArcA-P, but they vary across other bacterial species. A few other

mechanisms in E. coli that variously regulate initiation are DDAH

(datA-Dependent DnaA Hydrolysis, which is also regulated by IHF),

inhibition of the dnaA gene (by the SeqA protein), and reactivation of

DnaA by the lipid membrane.

ELONGATION

Once priming is complete, DNA polymerase III holoenzyme is loaded

into the DNA and replication begins. The catalytic mechanism of

DNA polymerase III involves the use of two metal ions in the active

site, and a region in the active site that can discriminate between

deoxyribonucleotides and ribonucleotides. The metal ions are general

divalent cations that help the 3' OH initiate a nucleophilic attack onto

the alpha phosphate of the deoxyribonucleotide and orient and

stabilize the negatively charged triphosphate on the

deoxyribonucleotide. Nucleophilic attack by the 3' OH on the alpha

phosphate releases pyrophosphate, which is then subsequently

hydrolyzed
of the parent strands of DNA is 3' 5' while the other is 5' 3'. To solve

this, replication occurs in opposite directions. Heading towards the

replication fork, the leading strand is synthesized in a continuous

fashion, only requiring one primer. On the other hand, the lagging

strand, heading away from the replication fork, is synthesized in a

series of short fragments known as Okazaki fragments, consequently

requiring many primers. The RNA primers of Okazaki fragments are

subsequently degraded by RNase H and DNA Polymerase I

(exonuclease), and the gaps (or nicks) are filled with

deoxyribonucleotides and sealed by the enzyme ligase.

RATE OF REPLICATION

The rate of DNA replication in a living cell was first measured as the

rate of phage T4 DNA elongation in phage-infected E. coli. During

the period of exponential DNA increase at 37 °C, the rate was 749

nucleotides per second. The mutation rate per base pair per replication

during phage T4 DNA synthesis is 1.7 per 108

TERMINATION

Termination of DNA replication in E. coli is completed through the

use of termination sequences and the Tus protein. These sequences

allow the two replication forks to pass through in only one direction,

but not the other.

DNA replication initially produces two catenated or linked circular

DNA duplexes, each comprising one parental strand and one newly

synthesised strand (by nature of semiconservative replication). This

catenation can be visualised as two interlinked rings which cannot be

separated. Topoisomerase 2 in E. coli unlinks or decatenates the two

circular DNA duplexes by breaking the phosphodiester bonds present

in two successive nucleotides of either parent DNA or newly formed

DNA and thereafter the ligating activity ligates that broken DNA

strand and so the two DNA get formed.

ROLLING CIRCLE REPLICATION

This is seen in bacterial conjugation where the same circular template

DNA rotates and around it the new strand develops.

When conjugation is initiated by a signal the relaxes enzyme creates a

nick in one of the strands of the conjugative plasmid at the oriT.

Relaxes may work alone or in a complex of over a dozen proteins

known collectively as a relaxosome. In the F-plasmid system the

relaxes enzyme is called Tral and the relax some consists of Tral,

Tray, TraM and the integrated host factor IHF. The nicked strand, or

T-strand, is then unwound from the unbroken strand and transferred to

the recipient cell in a 5'-terminus to 3'-terminus direction. The

remaining strand is replicated either independent of conjugative

action (vegetative replication beginning at the oriV) or in concert with

conjugation (conjugative replication similar to the rolling circle

replication of lambda phage). Conjugative replication may require a

second nick before successful transfer can occur. A recent report

claims to have inhibited conjugation with chemicals that mimic an

intermediate step of this second nicking event.

DNA polymerase III holoenzyme is the primary enzyme complex

involved in prokaryotic DNA replication. It was discovered by

Thomas Kornberg (son of Arthur Kornberg) and Malcolm Gefter in

1970. The complex has high processivity (i.e. the number of

nucleotides added per binding event) and, specifically referring to the

replication of the E.coli genome, works in conjunction with four other

DNA polymerases (Pol I, Pol II, Pol IV, and Pol V). Being the

primary holoenzyme involved in replication activity, the DNA Pol III

holoenzyme also has proofreading capabilities that corrects

replication mistakes by means of exonuclease activity reading 3'5' and

synthesizing 5'3'. DNA Pol III is a component of the replisome, which

is located at the replication fork.

COMPONENTS

The replisome is composed of the following:

➤ 2 DNA Pol III enzymes, each comprising a, & and 0 subunits. (It

has been proven that there is a third copy of Pol III at the replisome.

➤ the a subunit (encoded by the dnae gene) has the polymerase

activity.

➤ the & subunit (dnaQ) has 3'5' exonuclease activity.

➤ the 0 subunit (holE) stimulates the e subunit's proofreading.

➤ 2 ẞ units (dnaN) which act as sliding DNA clamps, they keep the

polymerase bound to the DNA.

➤ 1 y unit (also dnax) which acts as a clamp loader for the lagging

strand Okazaki fragments, helping the two ẞ subunits to form a unit

and bind to DNA. The y unit is made up of 5 y subunits which include

3 ү subunits, 1 8 subunit (holA), and 1 8' subunit (holB). The 8 is

involved in copying of the lagging strand.

➤ X (holC) and (hold) which form a 1:1 complex and bind to y or τ.

X can also mediate the switch from RNA primer to DNA.

DNA REPLICATION

In molecular biology, DNA replication is the biological process of

producing two identical replicas of DNA from one original DNA

molecule. DNA replication occurs in all living organisms acting as the

most essential part of biological inheritance. This is essential for cell

division during growth and repair of damaged tissues, while it also

ensures that each of the new cells receives its own copy of the DNA.

The cell possesses the distinctive property of division, which makes

replication of DNA essential.

DNA is made up of a double helix of two complementary strands. The

double helix describes the appearance of a double-stranded DNA

which is thus composed of two linear strands that run opposite to each

other and twist together to form.

During replication, these strands are separated. Each strand of the

original DNA molecule then serves as a template for the production of

its counterpart, a process referred to as semiconservative replication.

As a result of semi-conservative replication, the new helix will be

composed of
an original DNA strand as well as a newly synthesized strand.

Cellular proofreading and error-checking mechanisms ensure near

perfect fidelity for DNA replication.

In a cell, DNA replication begins at specific locations, or origins of

replication, in the genome which contains the genetic material of an

organism. Unwinding of DNA at the origin and synthesis of new

strands, accommodated by an enzyme known as helicase, results in

replication forks growing bi-directionally from the origin. A number

of proteins are associated with the replication fork to help in the

initiation and continuation of DNA synthesis. Most prominently,

DNA polymerase synthesizes the new strands by adding nucleotides

that complement each (template) strand. DNA replication occurs

during the S- stage of interphase.

DNA replication (DNA amplification) can also be performed in vitro

(artificially, outside a cell). DNA polymerases isolated from cells and

artificial DNA primers can be used to start DNA synthesis at known

sequences in a template DNA molecule.

Polymerase chain reaction (PCR), ligase chain reaction (LCR), and

transcription-mediated amplification (TMA) are examples. In March

2021, researchers reported evidence suggesting that a preliminary

form of transfer RNA, a necessary component of translation, the

biological synthesis of new proteins in accordance with the genetic

code, could have been a replicator molecule itself in the very early

development of life, or abiogenesis.

DNA STRUCTURE

DNA exists as a double-stranded structure, with both strands coiled

together to form the characteristic double helix. Each single strand of

DNA is a chain of four types of nucleotides. Nucleotides in DNA

contain a deoxyribose sugar, a phosphate, and a nucleobase. The four

types of nucleotide correspond four nucleobases adenine, cytosine,

guanine, to the and thymine, commonly abbreviated as A, C, G, and

T. Adenine and guanine are purine bases, while cytosine and thymine

are pyrimidines. These nucleotides form phosphodiester bonds,

creating the phosphate-deoxyribose backbone of the DNA double

helix with the nucleobases pointing inward (i.e., toward the opposing

strand). Nucleobases are matched between strands through hydrogen

bonds to form base pairs. Adenine pairs with thymine (two hydrogen

bonds), and guanine pairs with cytosine (three hydrogen bonds).

DNA strands have a directionality, and the different ends of a single

strand are called the "3' (three-prime) end" and the "5' (five-prime)

end". By convention, if the base sequence of a single strand of DNA

is given, the left end of the sequence is the 5' end, while the right end

of the sequence is the 3' end. The strands of the double helix are anti-

parallel, with one being 5' to 3', and the opposite strand 3' to 5'. These

terms refer to the carbon atom in deoxyribose to which the next

phosphate in the chain attaches. Directionality has consequences in

DNA synthesis, because DNA polymerase can synthesize DNA in

only one direction by adding nucleotides to the 3' end of a DNA

strand.

The pairing of complementary bases in DNA (through hydrogen

bonding) means that the information contained within each strand is

redundant. Phosphodiester (intra-strand) bonds are stronger than

hydrogen (inter- strand) bonds. The actual job of the phosphodiester

bonds is where in DNA polymers connect the 5' carbon atom of one

nucleotide to the 3' carbon atom of another nucleotide, while the

hydrogen bonds stabilize DNA double helices across the helix axis

but not in the direction of the axis. This makes it possible to separate

the strands from one another.

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