Section ESection E - - DNA DNA ReplicationReplication

E1 DNA Replication: an overviewE1 DNA Replication: an overview Semi-conservative mechanism, Replicons, Origins and ter

mini, Semi-discontinuous replication, RNA priming

E2 Bacterial DNA replication E2 Bacterial DNA replication Experimental systems, Initiation, Unwinding, Elongation,

Termination and segregation

E3 The cell cycle E3 The cell cycle The cell cycle, Cell cycle phases,

Checkpoints and their regulation, Cyclins and cyclin-dependent kinases, Regulation by E2F and Rb

E4 Eukaryotic DNA replicatiomE4 Eukaryotic DNA replicatiom Experimental systems, Origins and initiation,

Replication forks, Nuclear matrix, Telomere replication

ContentsContents

E1 DNA Replication: an overview — E1 DNA Replication: an overview —

Semi-conservative mechanismSemi-conservative mechanism

•A summary of the three postulated methods of DNA synthesis

•The Meselson - Stahl Experiment

E1 DNA Replication: an overview — E1 DNA Replication: an overview — Replicons, Origins and terminiReplicons, 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.

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.

• 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.

•The long, linear DNA molecules of eukaryotic chromosomes consist of multiple regions, each with its own origin.

•A typical mammalian cell has 50000-100000 replicons with a size range of 40-200 kb. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required.

Multiple eukaryotic replicons and repMultiple eukaryotic replicons and replication bubbleslication bubbles

replication bubbles replication fork

E1 DNA Replication: an overview — E1 DNA Replication: an overview —

Semi-discontinuous replicationSemi-discontinuous replication

•Many enzymes are involved in the DNA replication fork.

Discovery of Okazaki fragmentsDiscovery of Okazaki fragmentsEvidence for semi-discontinuous replicationEvidence for semi-discontinuous replication

[3H] thymidine pulse-chase labeling experiment1. Grow E. coli2. Add [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 (1000-2000 nt) = Okazaki fragments

3. Grow the cell in regular medium then analyze the small fragments join into high molecular weight DNA = Ligation of the Okazaki fragments

E1 DNA Replication: an overview — E1 DNA Replication: an overview — RNA RNA primingpriming

• 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

E2 Bacterial DNA replication — E2 Bacterial DNA replication —

Experimental systemsExperimental systems

1. Purified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (fX174, 5 Kb)

2. All the proteins and other factors for its complete replications

In vitro system: Put DNA and protein together to ask for replication question

E2 Bacterial DNA replication — E2 Bacterial DNA replication — InitiationInitiation

Re-initiation of bacterial replication at new origins before completion of the first round of replication

Study system: the E. coli origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes which behave like E. coli chromosome.

1. 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.

2. DnaA forms a complex of 30-40 molecules, facilitating melting of three 13 bp AT-rich repeat sequence for DnaB binding.

3. DnaB is a helicase that use the energy of ATP hydrolysis to further melt the double-stranded DNA .

4. Ssb (single-stranded binding protein) coats the unwinded DNA.

5. DNA primase load to synthesizes a short RNA primer for synthesis of the leading strand.

6. Primosome: DnaB helicase and DNA primase

E2 Bacterial DNA replication — E2 Bacterial DNA replication — UnwindingUnwinding

• Positive supercoiling: caused by removal of helical turns at the replication fork.

• Resolved by a type II topoisomerase called DNA gyrase

E2 Bacterial DNA replication — E2 Bacterial DNA replication — ElongationElongation

RNA primers

5‘3‘

3‘5‘

3‘5‘

3‘5‘

3‘5‘

5‘3‘

5‘3‘

5‘3‘

Primer removalPrimer removal

Gap fillingGap filling

LigationLigation

DNA polymerase III holoenzyme: 1. a dimer complex, one half synthesizing the leading stran

d and the other lagging strand.2. Having two polymerases in a single complex ensures th

at both strands are synthesized at the same rate3. Both polymerases contain an a-subunit---polymerase

e-subunit---3’5’ proofreading exonuclease b-subunit---clamp the polymerase to DNA

other 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.

Other two enzymes during elongation

1. Removal of RNA primer, and gap filling with DNA pol I

2. Ligation of Okazaki fragments are linked by DNA ligase.

E2 Bacterial DNA replication — E2 Bacterial DNA replication —

Termination and segregationTermination and segregation

•Terminus: containing several terminator sites (ter) approximately 180o opposite oirC.•Tus protein: ter binding protein, an inhibitor of the DnaB helicase

•Topoisomerase IV: a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.

Segregation

E3 The cell cycle — E3 The cell cycle — The cell cycleThe cell cycle

The cell cycle, or cell-division cycle, is the series of events that take place in a cell leading to its replication.

E3 The cell cycle — E3 The cell cycle — Cell cycle phasesCell cycle phases

G1 preparing for DNA replication (cell

growth)S

DNA replicationG2

a short gap before mitosisM

mitosis and cell division

E3 The cell cycle —E3 The cell cycle —

Checkpoints and their regulationCheckpoints 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.

mitogens

(+)

(-)

E3 The cell cycle — E3 The cell cycle — Cyclins and cyclin-dependent kinasesCyclins 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)

E3 The cell cycle —E3 The cell cycle — Regulation by E2F and RbRegulation 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.

E4 Eukaryotic DNA replication — E4 Eukaryotic DNA replication —

Experimental systemsExperimental systems

1. 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 system

3. Cell-free extract prepared from Xenopus (frog) eggs containing high concentration of replication proteins and can support in vitro replication.

E4 Eukaryotic DNA replication — E4 Eukaryotic DNA replication —

Origins and initiationOrigins and initiation

1. Clusters of about 20-50 replicons initiate simultaneously at defined times throughout S-phase

• Early S-phase: euchromatin replication• Late S-phase: heterochromatin replication• Centromeric and telomeric DNA replicate last

2. Only initiate once per cell cycle• Licensing factor: • required for initiation and inactivated after use• Can only enter into nucleus when the nuclear

envelope dissolves at mitosis

Licensing factor

Initiation

Initiation: origin

1. Yeast 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.

2. ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication.

E4 Eukaryotic DNA replication — E4 Eukaryotic DNA replication —

Replication forksReplication 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.

E4 Eukaryotic DNA replicatiom — E4 Eukaryotic DNA replicatiom — Nuclear matrixNuclear matrix

•A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription

Replication factories: all the replication enzymes, DNA associated with the replication forks in replication

BUdR labeling of DNA

Visualizing by immunoflurescence using BUdR antiboby

E4 Eukaryotic DNA replication — E4 Eukaryotic DNA replication —

Telomere replicationTelomere replication

1. Contains a short RNA molecule as telomeric DNA synthesis template

2. Telomerase 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)

3. The unlimited proliferative capacity of many cancer cells is associated with high telomerase activity.

Telomerase

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 chain

Processive DNA polymerases have 3’5’ exonuclease activity

Solving the problem of lagging strand synthesis -- Chromosomal ends shortening

5’ 3’5’3’

3’ 5’3’5’

5’ 3’5’3’Parental DNA

Daughter DNAs

telomerase

Elongation: three different DNA polymerases are involved.

1. 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.

2. DNA pol d: on the leading strand that replaces DNA pol a. can synthesize long DNA

3. DNA 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.

Crystal structure of phage T7 DNA polymerase

Exonuclease domain

template

Multiple choice Multiple choice questionsquestions

1 ． The number of replicons in a typical mammalian cell is .

A 40-200. B 400. C 1000-2000. D 50000-100000. 2. In prokaryotes,the lagging strand primers are remov

ed by . A 3' to 5' exonuclease. B DNA ligase. C DNA polymerase I. D DNA polymerase III.

3. The essential initiator protein at the E. coli origin of replication is .

A DnaA. B DnaB. C DnaC. D DnaE. 4. Which phase would a cell enter if it was starved of m

itogens before the R point? A G1. B S. C G2. D G0.

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 r

equirement for entry into S phase . C phosphorylation of E2F by a G1 cyclin-CDK complex is a critical req

uirement 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.

7. Prokaryotic plasmids can replicate in yeast cells if they contain a cloned yeast .

A ORC.

B CDK.

C ARS.

D RNA.

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