Section E DNA Replication

•Molecular Biology Course

E3: Eukaryotic DNA replicationE3: Eukaryotic DNA replication

•Molecular Biology Course

E1: DNA Replication: An OverviewE1: DNA Replication: An OverviewReplicons, semi-conservative, semi-discontinous, RNA priming

E2: Bacterial DNA replicationE2: Bacterial DNA replicationExperimental system, initiation, unwinding, elongation, termination & segregation

Experimental system, cell cycle, initiation, replication forks, nuclear matrix, telomere repl.

•DNA replication

E1: DNA Replication: An

Overview

E1: DNA Replication: An

Overview1.Replicons 2.semi-conservative mechanism3.semi-discontinous replication4.RNA priming

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

•DNA replication

E1-1 Replicons

Prokaryotic genome: a single circular DNA = a single replicon

Eukaryotic genome: multiple linear chromosomes & multiple replicons on each chromosome

•DNA 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.

•DNA replication

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.

•DNA replication

The long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions, each with its own orgin.

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 replication bubbles

•DNA replication

•DNA replication

replication bubbles replication fork

See Page 74 of your text book

•DNA replication

E1-2 Replication is Semi-conservative

Semi-conservative mechanism

15N labeling experiment

1. 15N labeling: grow cells in ??2. Collect DNA: grow cells in ??3. Separation: method ??4. Result interpretation

15N labeled DNA

unlabeled DN

A

•DNA replication

•DNA replication

E1-3 Replication is Semi-discontinuous

Semi-discontinuous replication

Ligation

•DNA replication

Okazaki fragments

Discovery of Okazaki fragmentsEvidence for semi-discontinuous

replication

[3H] thymidine pulse-chase labeling experiment1. Grow E. coli2. Add [3H] thymidine in the medium for a few secon

d 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

•DNA replication

•DNA replication

E1-4 RNA priming

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

•DNA replication

E2: Bacterial DNA replicationE2: Bacterial

DNA replication

1. Experimental system 2. initiation, 3. unwinding, 4. elongation, 5. termination & segregation

E2-1: In vitro experimental systems

1. Purified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (X174, 5 Kb)

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

•DNA replication

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

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

•DNA replication

E2-2: Initiation

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 DNA 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

Initiation

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

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

Resolved by a type II topoisomerase called DNA gyrase

•DNA replication

E2-3: Unwinding

•DNA replication

E2-4: Elongation

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

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

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

-subunit---3’5’ proofreading exonuclease -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.

Elongation: lagging strand replication

Polymerase III holoenzyme(DNA pol III)

DNA pol I (5’3’ exonulclease activity)

DNA pol I (5’3’ polymerase activity)

DNA ligase

•DNA replication

E2-5: Termination and Segregation

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

Termination

ter

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

Segregation

•DNA replication

E3: Eukaryotic DNA replication

E3: Eukaryotic DNA replication

1. Experimental system2. cell cycle, 3. initiation, 4. replication forks, 5. nuclear matrix, 6. telomere replication.

E3-1: In vitro experimental systems

1. Purified DNA : 2. All the proteins and other factors for its

complete replications

•DNA replication

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.

E3-2: Cell cycle When to replicate

•DNA replication

G1 preparing for DNA

replication (cell growth)S

DNA replicationG2

a short gap before mitosisM

mitosis and cell division

Cell cycle

Entry into the S-phase:Cyclins

Cyclin-dependent protein kinases (CDKs)

signaling

E3-2: Iniation of multiple replicons

•DNA replication

1. Timing2. Order

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 cycleLicensing 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.

E3-3: Replication fork & elongation

•DNA replication

1. unwinding2. enzymes

Replication fork

Unwinding DNA from parental nucleosomes before replication : 50 bp/sec, helicases and RP-A

New nucleosomes are assembled to DNA from a mixture of old and newly synthesized histones after the fork passes.

Elongation: three different DNA polymerases are involved.

1. DNA pol : 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 : on the leading strand that replaces DNA pol . can synthesize long DNA

3. DNA pol : on the lagging strand that replaces DNA pol synthesized Okazaki fragments are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.

E3-4: Nuclear Matrix

•DNA replication

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

E3-3: Telomere replication

•DNA replication

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

•DNA 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 replication

See movie for cancer metastasis

DNA polymerase control the fidelity of DNA replication

Proofreading 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 chain

Processive DNA polymerases have 3’5’ exonuclease activity

Supplemental 1

byE. coli polymerase

Proofreading

Supplemental 2

Crystal structure of phage T7 DNA polymerase

Exonuclease domain

template