Presentation on theme: "Section E DNA Replication"— Presentation transcript:
1 Section E DNA Replication Molecular Biology CourseSection EDNA Replication
2 E1: DNA Replication: An Overview Molecular Biology CourseE1: DNA Replication: An OverviewReplicons, semi-conservative, semi-discontinous, RNA primingE2: Bacterial DNA replicationExperimental system, initiation, unwinding, elongation, termination & segregationE3: Eukaryotic DNA replicationExperimental system, cell cycle, initiation, replication forks, nuclear matrix, telomere repl.
3 E1: DNA Replication: An Overview Repliconssemi-conservative mechanismsemi-discontinous replicationRNA priming
4 DNA replicationE1-1 RepliconsReplicon is any piece of DNA which replicates as a single unit. It contains an origin and sometimes a terminusOrigin is the DNA sequence where a replicon initiates its replication.Terminus is the DNA sequence where a replicon usually stops its replication
5 Prokaryotic genome: a single circular DNA = a single replicon DNA replicationProkaryotic genome: a single circular DNA = a single repliconEukaryotic genome: multiple linear chromosomes & multiple replicons on each chromosome
6 Bidirectional replication of a circular bacterial replicon DNA replicationBidirectional replication of a circular bacterial repliconAll prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circlular and comprise single replicons.There is a single termination site roughly 180o opposite the unique origin.
7 DNA replicationLinear viral DNA molecules usually have a single origin, replication details (see Section R)In all the cases, the origin is a complex region where the initiation of DNA replication and the control of the growth cycle of the organism are regulated and co-ordinated.
8 Multiple eukaryotic replicons and replication bubbles DNA replicationMultiple eukaryotic replicons and replication bubblesThe long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions, each with its own orgin.A typical mammalian cell has replicons with a size range of kb. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required
9 replication bubbles replication fork DNA replicationreplication bubbles replication forkSee Page 74 of your text book
10 E1-2 Replication is Semi-conservative DNA replicationE1-2 Replication is Semi-conservative
11 15N labeling experiment Semi-conservative mechanism DNA replicationSemi-conservative mechanism15N labeled DNAunlabeled DNA15N labeling experiment15N labeling: grow cells in ??Collect DNA: grow cells in ??Separation: method ??Result interpretation
13 E1-3 Replication is Semi-discontinuous DNA replicationE1-3 Replication is Semi-discontinuous
14 Semi-discontinuous replication DNA replicationSemi-discontinuous replicationLigationOkazaki fragments
15 Discovery of Okazaki fragments DNA replicationDiscovery of Okazaki fragmentsEvidence for semi-discontinuous replication[3H] thymidine pulse-chase labeling experimentGrow E. coliAdd [3H] thymidine in the medium for a few second spin down and break the cell to stop labeling analyze found a large fraction of nascent DNA ( nt) = Okazaki fragmentsGrow the cell in regular medium then analyze the small fragments join into high molecular weight DNA = Ligation of the Okazaki fragments
16 DNA replicationE1-4 RNA primingThe first few nucleotides at the 5’-end of Okazaki fragments are ribonucleotides. Hence, DNA synthesis is primed by RNA that is then removed before fragments are joined. Crucial for high fidelity of replication
17 E2: Bacterial DNA replication Experimental systeminitiation,unwinding,elongation,termination & segregation
18 E2-1: In vitro experimental systems DNA replicationE2-1: In vitro experimental systemsPurified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (fX174, 5 Kb)All the proteins and other factors for its complete replicationsIn vitro system: Put DNA and protein together to ask for replication question
19 DNA replicationE2-2: InitiationStudy system: the E. coli origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes which behave like E. coli chromosome.
20 oriC contains four 9 bp binding sites for the initiator protein DnaA oriC contains four 9 bp binding sites for the initiator protein DnaA. Synthesis of DnaA is coupled to growth rate so that initiation of replication is also coupled to growth rate.DnaA forms a complex of molecules, facilitating melting of three 13 bp AT-rich repeat sequence for DnaB binding.DnaB is a helicase that use the energy of DNA hydrolysis to further melt the double-stranded DNA .Ssb (single-stranded binding protein) coats the unwinded DNA.DNA primase load to synthesizes a short RNA primer for synthesis of the leading strand.Primosome: DnaB helicase and DNA primase
22 Re-initiation of bacterial replication at new origins before completion of the first round of replication
23 Resolved by a type II topoisomerase called DNA gyrase DNA replicationE2-3: UnwindingPositive supercoiling: caused by removal of helical turns at the replication fork.Resolved by a type II topoisomerase called DNA gyrase
25 DNA polymerase III holoenzyme: a dimer complex, one half synthesizing the leading strand and the other lagging strand.Having two polymerases in a single complex ensures that both strands are synthesized at the same rateBoth polymerases contain an a-subunit---polymerasee-subunit---3’5’ proofreading exonucleaseb-subunit---clamp the polymerase to DNAother subunits are different.Replisome: in vivo, DNA polymerase holoenzyme dimer, primosome (helicase) are physically associated in a large complex to synthesize DNA at a rate of 900 bp/sec.
33 E3-1: In vitro experimental systems DNA replicationE3-1: In vitro experimental systemsPurified DNA :All the proteins and other factors for its complete replications
34 Small animal viruses (simian virus 40, 5 kb) are good mammalian models for elongation (replication fork) but not for initiation.2. Yeast (Saccharomyces cerevisiae): 1.4 X 107 bp in 16 chromosomes, 400 replicons, much simpler than mammalian system and can serve as a model system3. Cell-free extract prepared from Xenopus (frog) eggs containing high concentration of replication proteins and can support in vitro replication.
35 E3-2: Cell cycle When to replicate DNA replicationE3-2: Cell cycle When to replicate
36 Cell cycle G1 preparing for DNA replication (cell growth) S a short gap before mitosisMmitosis and cell divisionEntry into the S-phase:CyclinsCyclin-dependent protein kinases (CDKs)signaling
37 E3-2: Iniation of multiple replicons DNA replicationE3-2: Iniation of multiple repliconsTimingOrder
38 Clusters of about 20-50 replicons initiate simultaneously at defined times throughout S-phase Early S-phase: euchromatin replicationLate S-phase: heterochromatin replicationCentromeric and telomeric DNA replicate last
39 2. Only initiate once per cell cycle Licensing factor:required for initiation and inactivated after useCan only enter into nucleus when the nuclear envelope dissolves at mitosis
41 Initiation: originYeast replication origins (ARS- autonomously replicating sequences, enables the prokaryotic plasmids to replicate in yeast).Minimal sequence of ARS: 11 bp [A/T]TTTAT[A/G]TTT[A/T] (TATA box)Additional copies of the above sequence is required for optimal efficiency.ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication.
43 Replication forkUnwinding DNA from parental nucleosomes before replication : 50 bp/sec, helicases and RP-ANew nucleosomes are assembled to DNA from a mixture of old and newly synthesized histones after the fork passes.
44 Elongation: three different DNA polymerases are involved. DNA pol a: contains primase activity and synthesizes RNA primers for the leading strands and each lagging strand fragments. Continues elongation with DNA but is replaced by the other two polymerases quickly.DNA pol d: on the leading strand that replaces DNA pol a. can synthesize long DNADNA pol e: on the lagging strand that replaces DNA pol a. synthesized Okazaki fragments are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.
45 DNA replicationE3-4: Nuclear MatrixA scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription
46 Replication factories: all the replication enzymes, DNA associated with the replication forks in replicationBUdR labeling of DNAVisualizing by immunoflurescence using BUdR antiboby
47 E3-3: Telomere replication DNA replicationE3-3: Telomere replicationSolving the problem of lagging strand synthesis-- Chromosomal ends shortening5’3’Parental DNA3’5’3’5’5’3’Daughter DNAs5’3’3’5’
49 DNA replicationTelomeraseContains a short RNA molecule as telomeric DNA synthesis templateTelomerase activity is repressed in the somatic cells of multicellular organism, resulting in a gradual shortening of the chromosomes with each cell generation, and ultimately cell death (related to cell aging)The unlimited proliferative capacity of many cancer cells is associated with high telomerase activity.See movie for cancer metastasis
50 Supplemental 1DNA polymerase control the fidelity of DNA replicationProofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chainProcessive DNA polymerases have 3’5’ exonuclease activity