DNA replication. DNA replication. replication model. replication fork. chapter 6

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1 DN chapter 6 DN two complementary s bases joined by hydrogen bonds separation of s each - template determines order of nucleotides in duplicate parent DN s separate two identical daughter s model dispersive model conservative model semiconservative model origins of parent DN parent DN origin of first bubble fork second two DN daughter molecules

2 unwinding and priming helicases unwind DN single- binding proteins hold nucleotides apart begins building a new polymerases build new s Overview Origin of antiparallel arrangement nucleotides only added to the 3 end semidiscontinous leading continuously synthesized lagging in pieces Primer Overall directions of fork DN polymerase DN ligase DN polymerases build new s leading lagging okazaki fragments connected by DN ligase also repairs mistakes or damage adds RN primer to begin process enzyme recap initiation of leading lagging direction of

3 fork overview Overview Origin of Overall directions of lagging lagging working backwards continues until it reaches previous fragment Leading Overview Origin of Overall directions of Parental DN Leading DN pol III Primer Primase Primer DN pol III Lagging 4 DN pol I DN ligase 3 emplate RN primer for fragment Okazaki fragment Okazaki fragment RN primer for fragment DN polymerase replisome DN polymerase III (x) β-clamp - sliding clamp DN helicase trombone model Parental DN onnecting protein DN pol III Helicase Leading DN pol III Lagging template Overall direction of

4 proofreading Nuclease DN polymerase DN ligase DN polymerase I proofreads newly made DN, replacing any incorrect nucleotides mismatch repair repair enzymes correct errors in base pairing nucleotide excision repair nuclease cuts out and replaces damaged stretches of DN error rate low mistakes may be passed on (mutations) eukaryotic in eukaryotes not as well understood begins at many sites yeast origins of autonomous replicating sequences about 400 RSs multiprotein origin complex (OR) regulation of origins change states licensing factors activation factors eukaryotic regulation of. OR complex binds to RS. licensing factors bind to OR pre- complex (pre-r) Mcm-Mcm7 - unwinds DN 3. activation of pre-r by DK 4. Mcm proteins act as helicase, move with fork 5. after, Mcm proteins displaced 3 4 licensing factors bind DK/DDK activate pre- R helicase activity RS OR licensing factors eukaryotic eukaryotic fork 5 classic DN polymerases α, β, ɣ, δ, ε PN - sliding clamp RP - holds s apart FEN- / ligase ligase FEN- RF LEDIN PN DN polymerase α LIN DN polymerase δ DN polymerase ε RP helica topoisomerase primase

5 chromatin structure nucleosomes assembly occurs rapidly histones remain intact during old and new histones distributed randomly DN repair Repair of mistakes essential DN susceptible to environmental damage fidelity of DN related to three distinct activities: accurate selection of nucleotides immediate proofreading post-replicative mismatch repair DN repair nucleotide excision repair (NER) removes bulky lesions pyrimidine dimers two pathways transcription-coupled global genomic steps recognition s separated lesion excised by endonucleases DN polymerase fills in gap ligase seals s DN repair base excision repair DN glycosylase - recognizes problem removes base P endonuclease / DN polymerase β removes deoxyribose phosphate DN polymerase β adds correct nucleotide DN ligase III reconnects

6 DN repair mismatch repair mismatch results in distortion of double helix parental must be identified E. coli - identified by methylated adenine residues in sequence on parental MutS - recognizes mismatch eukaryotes - not known H 3 H 3 Old New Mismatched MutS, MutL, MutH base H 3 H H 3 L S H 3 L H 3 H 3 H 3 S MutS, MutL, helicase, exonuclease DN polymerase and ligase DN repair double- breakage repair non-homologous end joining (NHEJ) lesion detected by Ku Ku recruits protein DN-PKS DN-PKS interacts with proteins which bring ends of DN together ends joined by DN ligase IV error prone - ends are lost homologous recombination has to occur soon after Ku DN-PKS DN ligase