Molecular BiologyThe Process

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)Department of Biochemistry,Medical College,Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190018. India.

Replication

• Process?

• Reaction?

• Enzymes?

• Replisome & Primosome?

• Helicases & Topoisomerases do?

• Starts at what?

• DNA Pol III vs DNA pol α/δ?

• Clamp in Pro- vs Eu- Karyotes?

Replication

Replication

• Replication in essentiality comprises of three stages/phases:

• Initiation

• Elongation

• Termination

Replication - Initiation

• Any DNA fragment that undergoes replication as a single unit is called as replicon

• Replication of DNA within a replicon by polymerases begins at one or more cis-acting sites termed as origin of replication

• The Origins increase the efficiency of initiation by providinga site for the assembly of the protein factors & enzymes required for replication

• Origins also provide a target for the regulation of replication and is the only phase in replication where regulation occurs

Replication - Initiation• Usually, in prokaryotes two replication forks proceed

bidirectionally away from the origin and the strands are copied as they separate until the terminus is reached

• All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circular and comprise single replicons

• Thus, there is a single termination site roughly 180o

opposite the unique origin

• In contrast, the long linear DNA molecules of eukaryotic chromosomes consist of multiple replicons, each with its own origin

• A typical mammalian cell has 50000 - 100000 replicons with a size range of 40-200 kb. Where replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA

Replication - Initiation• The E. coli bacterial replication origin is designate as oriC

and consists of essentially highly conserved 245 base pair DNA sequence, bearing a series of tandemly repeated elements (iterons)

• of four nonamers &

• three 13mers

• Nonamer has the consensus sequence of TTATCCACA

• 13mer has the consensus sequence of GATCTNTTNTTTT

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Replication - Initiation• At least nine different proteins or enzymes

participate in the initiation phase of replication

• The 9mer sequence is the binding site for the DnaA initiator protein

• A complex of 20-30 DnaA proteins first bind to four 9mer sequence in an ATP dependent manner causing the winding of DNA over them

• They facilitate the unwinding of DNA duplex by HU protein

• Then two hexamers of DnaB/DnaC complex binds to each of the unwound strand

• DnaB act as helicase causing further unwinding of DNA

• SSBP’s then bind to each strand to prevent the rewinding of the two strands and stabilize the two strands for the synthesis of DNA on them

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Replication - Regulation

• The initiation of replication is the only phase which is regulated

• One of the regulation mechanism is provided by the Dam methylase which methylates the N6 position of adenine in 5 -׳ GATC-3 ׳sequence

• The oriC of E. coli contains about 14 of these sequence

• If completely methylated in both strands; enables the replication to proceed

• In case of hemimethylated origins as occurs immediately after replication reinitiation does not occur

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Replication - Initiation• In yeast origins that span about 150bps and contain several

essential conserved sequences have been found and are called as Autonomously Replicating Sequences (ARS) or replicators

• The minimum length of DNA that supports the replication is only 11bp and has the consensus sequence of [A/T]TTTAT[A/G]TTT[A/T]

• The initiation of replication however requires a complex of multisubunit protein called as Origin Recognition Complex (ORC) to form an assembly of pre replicative complex (preRC) at ARS

• This begins at late M phase or early in G1 phase of cell cycle

• ORC is a hexamer of related proteins (Orc1-Orc6) which binds to the origin and then recruits two proteins Cdc6 and Cdt1 both members of cyclin dependent kinases

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Replication - Initiation

• Then six more proteins (Mcm2- Mcm7) bind to the origin forming a complex called as MCM (minichromosomemaintenance function) complex

• The whole assembly of 14 proteins is called as pre RC

• Pre RC is then activated to functional RC by the action of cyclindependent kinases (Cdk2, Cdk4/6, Ddk) by recruiting first Mcm10 and then causing the phosphorylation of five out of six MCM subunits

• Further replication initiation in eukaryotes is a multipoint process i.e., occurs at multiple ARS’s located all over the length of genome spaced 30,000 to 300,000bp apart

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Replication - Elongation• Since replication takes place in 5’→3’ direction thus only one

strand will be continuously synthesized called as leading strand (whose template has a 3’→5’ direction)

• The other strand called as lagging strand is synthesiseddiscontinuously in fragment 100-2000bp termed as okazakifragments which are then joined together by DNA ligase to form a continuous strand

• In fact, the physical direction of both leading and lagging strand synthesis is the same since the lagging strand is looped by 180o at the replication fork

• Elongation involves the synthesis of a RNA primer (of 10-60bases) on the template DNA stand which provides the hydroxyl group for the synthesis of DNA by the DNA polymerase

Replication - Elongation• In prokaryotes this is done by the primase (DnaG protein)

and later on replaced by Deoxynucleotides by the DNA polymerase III by its 3’ → 5’ exonuclease activity

• In eukaryotes this job is done by DNA polymerase α which has a integral primase activity, which continues elongation with DNA but is quickly replaced by DNA polymerase δ which is central enzyme of replication

• The synthesis of leading strand requires the primaseactivity only once but lagging strand requires the primer to be synthesised time & again at a distance of at least 2000 nucleotides on lagging strand this is done by primosomewhich consists of DnaB helicase & DnaG primase

• Primosome (in prokaryotes) remains associated with one of two core subuints of polymerase through out the synthesis of lagging strand

Replication - Termination• In E. coli the two replication forks meet almost 180o

opposite to oriC, around this region there are multiple copies of a 20bp sequence called as Ter which act as a trap for the replication fork

• Ter sequence are actually binding site for a protein called Tus(terminus utilization substance)

• Tus-Ter complex arrests the replication fork from only one direction (which one come first) and hence prevents over replication by the polymerase

• The final few hundred base pairs of DNA between the two arrested forks are then replicated producing two DNA circles which are topologically interlinked called as catenates

• Separation of catenates is then done by topoisomerase IV thus completing replication

Questions?