Replication occurs in three stages initiation, elongation, and termination,

Initiation:

⮚ In e.coli origin of replication has 245 Bp , the conserved sequence has 5 repeats of 9 Bp
sequence called R site . region rich in A-T sequence called the DNA unwinding element (DUE).
And has additional binding site called I site, IHF & FIS
⮚ At least 10 different types of protein/enzymes participate in initiation phase

⮚ They open DNA at origin and establish a prepriming complex (Place where DNA replication can
begin)
⮚ DnaA protein forms oligomers and hydrolyze ATP

⮚ The ATP-bound form is active and the ADP-bound form is inactive

⮚ 8 DnaA protein bound to ATP assemble to form a helical complex binding at R&I site , has higher
affinity towards R site

⮚ The binding of DNA around the complex induces a positive super coil this leads to the break of
A-T Rich DUE region
⮚ DnaC protein (an atp bound protein) loads DnaB protein on the separated strand
 ⮚ DnaC a hexamer binds to the hexamer of DnaB (is Helicase protein)
⮚ Two DnaB proteins are loaded in each strand of the Dna
⮚ The ATP present in DnaC is hydrolysed which releases DnaC
⮚ DnaB migrates along the DNA strand unwinding the DNA in opposite direction of each creating
two replication forks
⮚ As the strands get separated SSB proteins come and bind to the strand
⮚ DNA gyrase reduces the stress caused by unwinding of the strands

Elongation
➢ elongation includes synthesis of leading and lagging strand synthesis it involves many enzymes
➢ it begins with unwinding of the parent DNA strand by helicase , topoisomerases helps in
reducing the physical stress.SSB protein then bind to separated strands
➢ LEADING STRAND synthesis begins with the synthesis by primase (DnaG Protein) which adds a
RNA primer at the replication origin, primase interact with helicase to start the reaction
➢ This leads to the synthesis of primer, primer is synthesized opposite to that in which the DnaB
helicase is moving
➢ DnaB helicase moves along the strand that becomes the lagging strand in DNA synthesis
➢ Deoxyribonucleotides are added to this primer by a DNA polymerase III, leading strand
continuous with the unwinding of DNA
➢ LAGGING STRAND synthesis in short Okazaki fragments, strats by RNA primer synthesis followed
by , DNA polymerase III binds to the RNA primer and adds deoxyribonucleotides
➢ both the leading and lagging strand are synthesized by DNA polymerase III, primosome, a protein
complex containing DNA primase and DNA helicase
➢ DNA polymerase III uses one set of its core subunits to synthesize the leading strand
continuously, while the other set of core subunits to from one Okazaki fragment to the next on
the looped lagging strand
➢ DnaG primase occasionally associates with DnaB helicase and synthesizes a short RNA primer as
the unwinding of the DNA occurs
➢ The complete replication apparatus moving along the DNA molecule at a replication fork is called
the replisome. includes all the proteins
➢ The replication proteins are thus linked together into a single large unit enabling DNA to be
synthesized on both sides of the replication fork in a coordinated and efficient manner
➢ Beta sliding clamp is positioned at the primer which helps to keep the polymerase intact
➢ Beta sliding clamp gets dissociate from the polymerase When synthesis of an Okazaki fragment
has been completed this marks the complete synthesis of a single Okazaki fragment
➢ the beta loading complex binds to ATP and to the new Beta sliding clamp this binding opens the
ring hear enters the new lagging strand which has a primer to it
➢ the clamp loading complex hydrolyse the ATP
➢ The replisome promotes rapid DNA synthesis, adding -1,000 nucleotides per second in both the
leading and the lagging strands
➢ RNA primer are removed by 5’ —> 3’ exonuclease activity of DNA polymerase I and the nick are
sealed by DNA ligase

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