DNA Replication Tsung-Luo Jinn

Three stages of DNA Replication Initiation: primosome, a protein complex act of initiation Elongation: replisome, a protein complex, associate with particular DNA structure to unwind the DNA and synthesis daughter strands Termination: Tus protein and ter termination site

Conditional lethal mutants: Temperature-sensitive mutants --the dna genes Quick-stop mutants--the major class Defect in elongation Slow-stop mutants--the smaller class Defect in initiation

Function of DNA polymerase --the enzyme that can synthesis a new strand on a template strand In E.Coli DNA polymerase I: coded by polA DNA polymerase II: coded by polB DNA polymerase III:coded by polC --multisubunit In Phage --T 4,T 5, T 7,SPO1 Code for DNA polymerase

DNA polymerase I III II 5‘ 3‘ 5‘ 3‘ polymerase exonuclease ＋＋＋ ＋＋＋ ＋－－ Replicase －－＋ MW (kDa) Numbers ??? Bioactivity Gene pol C ＊ pol A pol B Summary In Bacteria

DNA polymerase δ replicase Priming replication repairing location α βγε Nu Mito repairing function replication In Eukaryotic 5‘-3’ plolymerization 3‘-5’ exonuclease ＋＋＋＋＋ －－＋＋＋

Errors in DNA synthesis Substitution Frameshifts --insertion,deletion --misspairing Determined by proofreading Affected by processivity Nonsense --early stop

The fidelity of DNA replication Control at two different stages: Control at the incoming base --presynthetic control Proofreading control * ~10 -6

polymerization --an 5‘ to 3’ elongation

The all DNA polymerase in Bacteria with 3’-5’ exonuclease activity---proofreading DNA polymerase I (103kDa) -- proteolytic treatment C-large fragment (68kDa) with polymerase and 3‘-5’ exonuclease activity --Klenow fragment N-small fragment(35kDa) --5’-3’ exonuclease (up to ~10 bases at a time)

The Nick translation DNA polymerase I --in Nicked DNA --in vitro Need: 5‘-3’ polymerase 5‘-3’ exonuclease 3‘-5’ exonuclease * Rolling circle

Priming reaction in replication Primase: RNA polymerase ~10 bases Nicked DNA 3‘-OH Terminal protein:Ser :dCMP-3’OH With different way

Right Hand structure of DNA polymerase B-form DNA A-form DNA

DNA replication A semidiscontinuous replication Leading strand: a continuous strand Lagging strand: a series fragments are jointed ~10-20 kb length Okazaki fragments

continuous discontinuous The semidiscontinuous replication

Primase,DNA Polymerase III DNA Polymerase I, DNA Ligase Primase:Code by dnaG --RNA polymerase bases primer for DNA synthesis --primer start with the sequence pppAG, in 3’-GTC-5’ in template DNA polymerase III DNA polymerase I DNA ligase * Mammalian system

Ligation ligase Phage--ATP E.coli--NAD Nicotinamide adenine dinucleotide *T 4 DNA ligase

Conversion of ψX 174 ds DNA into ssDNA in vitro TraY: bind to the oriT TraI: A protein,a relaxase, bind to TraY in the oriT Rep: helicase--unwinding DNA --ATP dependent SSB:single-strand DNA binding protein --Prevent DNA reform to ds DNA helicase

Two types of priming in E.Coli OriC and ψX systems OriC: the bacteria origin DnaB--helicase (5’-3’) ψX: the phage origin DnaB--helicase (5’-3’) require primosome see page398 to replace SSB

Problem in simultaneously synthesizing leading and lagging strands

DNA polymerase III subunits assembly

The dimer surround the duplex, providing the sliding clamp that allow the holoenzyme to slide along DNA

The replication fork Coordinating synthesis of leading and lagging strands DnaB:helicase DnaG:primase polymeaseIII τ leading strands lagging strands Increase synthesis speed Prevent polymerase fall off

Dissociation and Reassociation of βclamp during DNA replication

Tus protein and termination of replication at ter site Termination In E.Coli Ter with 23 bases consensus sequences Tus protein (36 kDa) Stop helicase to unwind DNA See page 354 Countra-helicase activity

Summary The protein component of replication complex

Initiation of replication fork at replication origin Ori C origin Ori λ origin In E.Coli

Initiation of replication Ori C in vitro DnaA, DnaB, DnaC, HU, Gyrase, SSB--primosome ATP needed A minimal origin --Kornberg, 1953 DnaG: primer--replication

Immunolocalization of replication complex at Ori C origin-by antibody against DnaB The DnaB:DnaC complex ~480kDa --- ~6nm

Initiation of replication Ori λ in vitro Replication is activated by genes O and P transcription—away from ori. O protein bind to ori to generate a spherical structure -- O-some(~11nm), and then interacted with P protein bind to the replication origin within geneO -- primosome--DNA bending O protein==DnaA P protein==DnaC DnaB, Gyrase, SSB, Dnak,DnaJ DNA replication, is trigger after P protein release DnaG: primer--replication

How to ensure initiation of replication only once per cycle ?

Methylated DNA in the origin, can be distinguished from the replicated DNA In OriC, 11 copies of GATC Adenine-N 6 -CH 3 by Dam methylase, before replication Daughter strands with hemimethylated DNA, can not be used to initiated a replication cycle Delay remethylation in oriC---delay replication

The methylated DNA

The model The membrane bound inhibitor binds to hemimethylated DNA Remethylated DNA, inhibitor releases DnaA binds to oriC --initiate replication

How to control the multiple replicons is activated only once time in a single cycle ?

By rate-limiting component which function only once at the origin--licensing factor Prevent more than one cycle of replication--by removing the component Two purposes Makes the replication initiation dependent on cell division HOW !!

The licensing factor in Yeast ORC: origin recognition complex, bind to A and B1 in ARS Cdc6, a highly unstable protein (half-life ＜ 5 min), synthesis only in the G1 phase Cdc6, allow Mcm bind to complex Replication initiation--Cdc6-Mcm are displaced