Post on 17-Jul-2015
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?