DNA REPLICATION

DNA REPLICATION ITS MECHANISM AND FACTORS THAT REGULATE THE DNA REPLICATION PROCESS

1. DNA
REPLICATION
RVS Chaitanya Koppala
Assistant Professor
Vignan Institute of Pharmaceutical Technology
Visakhapatnam

2. DNA REPLICATION
INTRODUCTION.
1. Type of DNAs.
2. Modes of DNA Replication.
3. Enzymes used in DNA Replication.
4. Stages of DNA Replication.
5. Comparison of Eukaryotic & Prokaryotic
6. Replication.

DNA is a double helical structure.
 There are two polynucleotide chain coiled about one another in a spiral.
 Each polynucleotide chain consists of a sequence of nucleotides linked together by
phosphodiester bonds.
 The two polynucleotide strands are held together in their helical configuration by
hydrogen bonding between bases in opposing strands.
 The base-pairing is specific; adenine is always paired with thymine, and
guanine is always paired with cytosine.
 The two strand of a DNA double helix are complementary (not identical).
 Complementarity of two strands, that makes DNA uniquely suited to store and
transmit genetic information.
 The base pair in DNA are stacked 3.4 Ǻ apart with 10 base pair per turn.
 The sugar-phosphate backbones of two complementary strands are antiparallel .
DNA REPLICATION
 Replication of DNA is central to all biology.
 DNA replication is a process to produce new DNA molecules that have the same
base sequence. Occurs during interphase of the cell cycle
 DNA replication is semi-conservative.
 The parent DNA strand separates into two Each strand serves as a template for new
complementary strands. The new double helix is half original.
 DNA molecule exhibit a considerable amount of conformational flexibility.
(It is different in different physiological conditions.)
 DNA has 3 major forms:-
1. B – form

3. 2. A - form
3. Z – form

4. Helicase: -
 Helicase uses energy from the ATP to break the hydrogen bond holding the base pair
together.
 This allow the two parental strands of DNA to begin unwinding and forms two
replication fork.
 Two different helicases are involved for unwinding of each strand.
 It is coded by gene DNA B.
RNA Primase: -
 Primase is an enzyme that copies a DNA template strand by making a RNA strand
complementary to it.
 Primase synthesizes a short (about 10 nucleotides) RNA primer in the 5’ to 3’ direction.
 It is product of DNA G gene.
 Primase activity requires the formation of a complex of primase and at least six proteins
(protein i, n, n’, n” plus the product of gene DNA B and DNA C), the complex is called
the primo some.
Single Stranded Binding Protein (SSBP): -
 Single-stranded DNA binding protein (SSB) binds to the single stranded portion of each
DNA strand, preventing the strands from reassociating and protecting them from
degradation
 By nucleases.
 The SSBP binds DNA as tetramer and their binding exhibit cooperativity (i.e. the
binding of one tetramer to adjacent segment of single stranded DNA).
 SSBP binded DNA replicate over hundred times faster than uncomplexed single
stranded DNA.
 It helps to stabilize the extended single stranded template needed for polymerization.
 It prevent folding back of DNA.
DNA Gyrase (Type II Topoisomerase):-
 This enzyme can change the state of supercoiling in a DNA molecules.
 It relieves the tension that builds up during replication.
 It catalyze the formation of negative supercoil in DNA.
 It removes positive supercoils.

5. DNA Polymerase I :-
 DNA polymerase I is involved in removing RNA primers in the processing of DNA
after replication.
 Both DNA polymerase I and III have the ability to "proofread" their work by
means of a 3' 5' exonuclease activity.
DNA Polymerase III :-
 DNA polymerase III is a complex enzyme containing 10 different polypeptides (a,e,ɣ
etc.) . All those polypeptides must be present for proper replicative function.
 The 5’ to 3’ polymerase activity and the 5’to 3’ exonuclease activity both present on
the a-polypeptides of DNA polymerase III.
 The 3’to 5’ proofreading activity of polymerase III present on the e -polypeptide.
 The function of other subunit still uncertain.
 It catalyse the chemical reactions for polymerization of nucleotides.
 DNA polymerase III begins synthesizing DNA in the
 5’3’direction, beginning at the 3’ end of each RNA primer.
Structure of DNA Polymerase III :-
 DNA polymerase resembles a hand that grip the primer-template junction.
 The speed of DNA synthesis is largely due to the processive nature of DNA polymerase.
 IN case of DNA polymerase , the degree of processivity is defined as the average no.
nucleotide added each time the enzyme binds a primertemplate junction.
 It can be from few nucleotides to more than 50000 bases added per binding event.

6.  DNA ligase catalyses covalent closure of the resulting singlestranded “nick”.
 DNA ligase joins Okazaki fragments, converting them to a continuous strand of
DNA.
Terminus Utilization Substance ( TUS) Protein:-
 It is a protein that binds to terminator sequences and acts as a counter-helicase when it
comes in contact with an advancing helicase.
 The bound Tus protein effectively halts DNA polymerase movement.
 Tus helps to end the DNA replication in prokaryotes.

 Initiation :-Occurs at the origin of replication.
 Elongation:-Involves the addition of new nucleotides based on complementarity of the
template strand.
 Termination:-Occurs at a specific termination site.

7.  At ore there is presence of specific base sequences regions.
 One is 9 base pair long regions which have four repeats and another is 13 base pair long
have 3 repeats also called 13 mer region.
 13 mers are A-T base pair rich regions.
 Dna –A proteins binds to 9 b.p. region, which cause change in 13 mer region and it start
open up. In 13 mer region due to presence of A-T b.p. ,it is easy to open as A-T b.p. has
two hydrogen bonds as compared to C-G b.p. as it contains three hydrogen bond.
 After this there is attachment of helicase and SSB proteins and other enzymes to carry
on DNA replication.

10. TERMINATION:-
 In prokaryotes DNA replication terminates when replication fork reach specific
‘termination site’.
 The arrest of DNA replication in Escherichia coli is triggered by the encounter of a
replisome with a Tus protein-Ter DNA complex.
 A replication fork can pass through a Tus-Ter complex when traveling in one direction
but not the other, and the chromosomal Ter sites are oriented so replication forks can
enter, but not exit, the terminus region.
 The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but
details of the mechanism are uncertain
 The two replication fork meet each other on the opposite end of the parental circular
DNA.

 It is done by exonuclease activity of DNA Pol. III . The role of exonuclease become
clear when it was determined that they have a strong preference to degrade DNA
containing mismatch base pair.
 The removal of mismatched nucleotide is facilitated by the reduced ability of DNA
polymerase to add nucleotide an incorrectly base paired primer.
 As for processive DNA synthesis , proofreading occurs without releasing the
 DNA from the polymerase.
 When a mismatched base pair is present in the polymerase active site , the primer :
template junction is destabilized , creating base pairs of unpaired DNA.
 The newly unpaired 3’end moves from the polymerase active site to exonuclease active
site.
 The incorrect nucleotide is removed by the exonuclease.

11.  The removal of mismatched base allow the primer :template junction to reform and
rebind to the polymerase active site , enabling DNA synthesis to continue.