2 Contents E1 DNA Replication: an overview E2 Bacterial DNA replication Semi-conservative mechanism, Replicons, Origins and termini, Semi-discontinuous replication, RNA primingE2 Bacterial DNA replicationExperimental systems, Initiation, Unwinding, Elongation, Termination and segregationE3 The cell cycleThe cell cycle, Cell cycle phases, Checkpoints and their regulation, Cyclins and cyclin-dependent kinases, Regulation by E2F and RbE4 Eukaryotic DNA replicatiomExperimental systems, Origins and initiation, Replication forks, Nuclear matrix, Telomere replication
3 E1 DNA Replication: an overview — Semi-conservative mechanism A summary of the three postulated methods of DNA synthesis
6 E1 DNA Replication: an overview — Replicons, Origins and termini Replicon is any piece of DNA which replicates as a single unit. It contains an origin and sometimes a terminus.Origin is the DNA sequence where a replicon initiates its replication.Terminus is the DNA sequence where a replicon usually stops its replication.
7 Bidirectional replication of a circular bacterial replicon All 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.
8 Linear 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.
9 Multiple eukaryotic replicons and replication bubbles The long, linear DNA molecules of eukaryotic chromosomes consist of multiple regions, each with its own origin.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.replication bubbles replication fork
10 E1 DNA Replication: an overview — Semi-discontinuous replication Many enzymes are involved in the DNA replication fork.
12 Discovery of Okazaki fragments Evidence 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
14 E1 DNA Replication: an overview — RNA priming The leading strand and all lagging strand fragment are primed by synthesis of a short piece of RNA which is then elongated with DNA. The primers are removed by DNA before ligation. The mechanism helps to maintain high replication fidelity.The 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
15 E2 Bacterial DNA replication — Experimental systems Purified 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
16 E2 Bacterial DNA replication — Initiation Re-initiation of bacterial replication at new origins before completion of the first round of replicationStudy system: the E. coli origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes which behave like E. coli chromosome.
18 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 ATP 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
19 E2 Bacterial DNA replication — Unwinding Positive supercoiling: caused by removal of helical turns at the replication fork.Resolved by a type II topoisomerase called DNA gyrase
21 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.
23 Other two enzymes during elongation 1. Removal of RNA primer, and gap filling with DNA pol I2. Ligation of Okazaki fragments are linked by DNA ligase.
24 E2 Bacterial DNA replication — Termination and segregation Terminus: containing several terminator sites (ter) approximately 180o opposite oirC.Tus protein: ter binding protein, an inhibitor of the DnaB helicase
25 SegregationTopoisomerase IV: a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.
26 E3 The cell cycle — The cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell leading to its replication.
27 E3 The cell cycle — Cell cycle phases G1preparing for DNA replication (cell growth)SDNA replicationG2a short gap before mitosisMmitosis and cell division
28 E3 The cell cycle — Checkpoints and their regulation The cell cycle is regulated in response to the cell’s environment and to avoid the proliferation of damaged cells.Checkpoint are stages at which the cell cycle may be halted if the circumstance are not right for cell division.
30 E3 The cell cycle — Cyclins and cyclin-dependent kinases The cell cycle is controlled through protein phosphorylation(磷酸化), which is catalysed(催化) by multiple protein kinase complexes.These complexes consist of cyclins(细胞周期调节蛋白), the regulatory subunits, and cyclin-dependent kinases( CDKs)
32 E3 The cell cycle — Regulation by E2F and Rb E2F family members play a major role during the G1/S transition in the mammalian cell cycle.Among E2F transcriptional targets are cyclins, CDKs, checkpoints regulators, DNA repair and replication proteins.The activity of E2F is inhibited by the binding of the protein Rb (the retinoblastoma tumor suppressor protein) and related proteins.
34 E4 Eukaryotic DNA replication — Experimental systems Small animal viruses (simian virus 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 E4 Eukaryotic DNA replication — Origins and initiation Clusters of about replicons initiate simultaneously at defined times throughout S-phaseEarly S-phase: euchromatin replicationLate S-phase: heterochromatin replicationCentromeric and telomeric DNA replicate last
36 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
38 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.
39 E4 Eukaryotic DNA replication — Replication forks The replication fork is a structure that forms within the nucleus during DNA replication.It is created by helicases(解螺旋酶), which break the hydrogen bonds holding the two DNA strands together.The resulting structure has two branching "prongs", each one made up of a single strand of DNA, that are called the leading and lagging strands. DNA polymerase creates new partners for the two strands by adding nucleotides.
41 E4 Eukaryotic DNA replicatiom — Nuclear matrix A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription
42 Replication factories: all the replication enzymes, DNA associated with the replication forks in replicationBUdR labeling of DNAVisualizing by immunoflurescence using BUdR antiboby
43 E4 Eukaryotic DNA replication — Telomere replication
44 TelomeraseContains 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.
45 DNA polymerase control the fidelity of DNA replication Proofreading refers to any mechanisms 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
46 Solving the problem of lagging strand synthesis -- Chromosomal ends shortening5’3’Parental DNA3’5’3’5’5’3’Daughter DNAs5’3’3’5’
48 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 which are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.
49 Crystal structure of phage T7 DNA polymerase templateExonuclease domain
50 Multiple choice questions 1．The number of replicons in a typical mammalian cell isAB 400.CD2. In prokaryotes,the lagging strand primers are removed byA 3' to 5' exonuclease.B DNA ligase.C DNA polymerase I.D DNA polymerase III.
51 3. The essential initiator protein at the E 3. The essential initiator protein at the E. coli origin of replication isA DnaA.B DnaB.C DnaC.D DnaE.4. Which phase would a cell enter if it was starved of mitogens before the R point?A G1.B S.C G2.D G0.
52 5. Which one of the following statements is true? A once the cell has passed the R point, cell division is inevitable.B the phosphorylation of Rb by a G1 cyclin-CDK complex is a critical requirement for entry into S phase .C phosphorylation of E2F by a G1 cyclin-CDK complex is a critical requirement for entry into S phase.D cyclin D1 and INK4 p16 are tumor suppressor proteins.6. In eukaryotes, euchromatin replicates predominantly .A in early S-phase.B in mid S-phase.C in late S-phase.D in G2-phase.
53 7. Prokaryotic plasmids can replicate in yeast cells if they contain a cloned yeast . A ORC.B CDK.C ARS.D RNA.