DNA replication

Semi-conservative mechanism

1958, Meselson & Stahl

15N labeling experiment

Rosalind Franklin (1920-1958) Maurice Wilkins (1916-2004)

Francis Crick (1916-2004)James Watson (1928-)

Discovery of DNA structure 1962 Nobel Prize

The substrates of DNA synthesis

dNTPs – dATP, dGTP, dCTP, dTTP

Direction: 5’-3’

OH

T C A

T C A C

OH 3’

ppp OH

C

+

+ ppi

5’PPP

5’PPP

5’ 3’

A T C G

+ 5’ ppp OH 3’

G

ppp OH

G A T C G

5’ ppp OH 3’

3’ 5’ ???

A T C G

+ 5’ ppp OH 3’

G

ppp OH

G

5’ ppp ppp OH 3’

A T C G

???

A T C G

ppp OH+ ppp

A

p OH

T C G

pp

p OH

T C G T C G

pp p OH

Proofreading???

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

All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circular and comprise single replicons.

There is a single termination site roughly 180o opposite the unique origin.

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

Semi-discontinuous replication

Experimental evidences [3H] thymidine pulse-chase labeling experiment

1. Grow E. coli2. Add [3H] thymidine in the medium for a few second, spin down an

d break the cell to stop labeling, analyze and find 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

Back

Bacterial DNA replication

Experimental systems

1. Purified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (ΦX174, 5 Kb)2. All the proteins and other factors for its complete replications

Study system

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

E.coli chromosome.

Initiation: oriC

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 attaches to the DNA and synthesizes a short RNA primer for synthesis of the leading strand.

6. Primosome DnaB helicase and DNA primase

Unwinding

Positive supercoiling: caused by removal of

helical turns at the replication fork.

Resolved by a type II topoisomerase called

DNA gyrase

Elongation

DNA polymerase III holoenzyme

1. A dimer complex, one half synthesizing the leading strand and the other lagging strand.

2. Having two polymerases in a single complex ensures that both strands are synthesized at the same rate

3. Both polymerases contain an α-subunit---polymerase ε-subunit---3’ 5’ proofreading exonuclease β-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.

Prokaryotic DNA replication

Termination and segregation

Terminus

containing several terminator sites (ter) approximately 180o opposite oriC.

Tus protein ter binding protein, an inhibitor of the DnaB helicase

Topoisomerase IV a type II DNA topoisomerase, function to unlink the interli

nked daughter genomes.

Eukaryotic DNA replication

Experimental 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): 14 Mb 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.

Cell cycle

Entry into the S-phase

Cyclins

CDKs (Cyclin-dependent protein kinases)

DNA Replication

DNA replication is semi-conservative, one strand serves as the template for the second strand. Furthermore, DNA replication only occurs at a specific step in the cell cycle. The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle.

Stage Activity Duration G1 Growth and increase in cell size 10 hr S DNA synthesis 8 hr G2 Post-DNA synthesis 5 hr M Mitosis 1 hr

DNA replication has two requirements that must be met:

1. DNA template 2. Free 3' -OH group

Origin 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 inactivated after use can only enter into nucleus when the nuclear envelope dissolves at mitosis

Electron Microscopy of replicating DNA revealsreplicating bubbles.

3. Individual yeast replication origins (ARS) have been cloned into prokaryotic plasmids which allow these plasmids to replicate in yeast (an eukaryote).

ARSs autonomously replicating sequences

Minimal sequence 11 bp

[A/T]TTTAT[A/G]TTT[A/T] (TATA box)

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

Elongation

1. Replication fork

- unwinding DNA from nucleosomes: 50 bp/sec

- need helicases and replication protein A (RP-A)

- new nucleosomes are assembled to DNA from a mixture

of old and newly synthesized histones after the fork passes

2. ElongationThree different DNA polymerases are involved

1) DNA pol α 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 δ on the leading strand that replaces DNA pol α., can synthesize long DNA

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

Nuclear matrix

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

2. Replication factories

containing all the replication

enzymes and DNA associated

with the replication forks

in replication

BudR labeling of DNA

Telomere replication

Telomerase

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 activity is repressed in somatic cells of multicelluar organisms resulting in a gradual shortening of the chromosome with each cell generation. As this shortening reaches informational DNA, the cells senesce and die.

cell division

informational DNA

cell dies or does not divide

When telomerase activity is repressed

Mutagenesis

Mutation

Permanent, heritable alterations in the base sequence of DNA

Reasons

1. Spontaneous errors in DNA replication or meiotic recombination

2. A consequence of the damaging effects of physical or chemical mutagens on DNA

Point mutationA singe base change: transition, transversion

The effects of point mutation

Phenotypic effectsNoncoding DNANonregulatory DNA Silent mutation No 3rd position of a codon

Coding DNA altered AA Missense mutation Yes or No

Coding DNA stop codon Nonsense mutation Yes Truncated protein

Insertions & deletions

The addition or loss of one or more bases in a DNA region

Frameshift mutations The ORF of a protein encoded gene is changed so that the

C-terminal side of the mutation is completely changed.

Genetic polymorphisms

Caused by accumulation of many silent and other nonlethal mutations

Replication fidelityImportant for preserve the genetic information from one generati

on to the next, spontaneous errors in DNA replication is very rare, e.g. one error per 1010 base in E. coli.

Molecular mechanisms for the replication fidelity1. DNA polymerase: Waston-Crick base pairing

2. 3’ 5’proofreading exonuclease.

3. RNA priming: proofreading the 5’end of the lagging strand

4. Mismatch repair

Mutagens

Causing DNA damage that can be converted to mutations.

Physical mutagens High-energy ionizing radiation X-rays and γ-rays strand breaks and base/sugar destruction Nonionizing radiation UV light pyrimidine dimers

Chemical mutagens Base analogs direct mutagenesis Nitrous acid deaminates C to produce U Alkylating agents Arylating agents indirect-lesion mutagenesis Intercalators: e.g. EB

MutagenesisThe molecular process in which the mutation is

generated.

Note the great majority of lesions introduced by chemical and physical mutagens are repaired by one or more of the error-free DNA repair mechanisms before the lesions is encounter by a replication fork

Direct mutagenesis The stable, unrepaired base with altered base pairing

properties in the DNA is fixed to a mutation during DNA replication.

Indirect mutagenesis The mutation is introduced as a result of an error-prone repair. Translesion DNA synthesis to maintain the DNA integrity but not the sequence accuracy

when damage occurs immediately ahead of an advancing for

k, which is unsuitable for recombination repair, the daughter

strand is synthesized regardless of the the base identity of the

damaged sites of the parental DNA.

DNA damage and repair

DNA lesionsOxidative damage1. Occurs undernormal condition2. Increased byionizing radiationphysical mutagens

AlkylationAlkylating agentsChemical mutagens

Bulky adductsUV lightphysical mutagensCarcinogenChemical mutagens

Biological effects of the unrepaired DNA lesions

Physical distortion ofthe local DNA structure

Blocks replicationand/or transcription

Lethal

Altered chemistry of the bases

Allowed to Remain in the DNA

A mutation could become fixed by direct or indirect mutagenesis

Mutagenic

Spontaneous DNA lesions

1. Inherent chemical reactivity of the DNA

2. The presence of normal, reactive chemical species within the cell

- Deamination C U methylcytosine T

- Depurination break of the glycosylic bond, non-coding lesion

- Depyrimidine

Oxidative damage

1. occurs under NORMAL conditions in all aerobic cells due to the presence of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and the hydroxyl radicals (•OH).

2. The level of this damage can be INCREEASED by hydroxyl radicals from the radiolysis of H2O caused by ionizing radiation

Alkylation

1. Electrophilic chemicals adds alkyl groups to various positions on nucleic acids

2. Distinct from those methylated by normal methylating enzymes.

3. Typical alkylating agents:

MMS methylmethane sulfonate

EMS ethylmethane sulfonate

ENU ethylnitrosourea

Bulky adducts

1. DNA lesions that distort the double helix and cause localized denaturation, for example pyrimidine dimers

arylating agents adducts

2. These lesions disrupt the normal function of the DNA

DNA repair

Photoreactivation

1. Monomerization of cyclobutane pyrimidine dimers by DNA photolyases in the presence of visible light

2. Direct reversal of a lesion and is error-free

Alkyltransferase

1. Removing the alkyl group from mutagenic O6-alkylguanine which can base-pair with T. The alkyl group is transferred to the protein itself and inactivate it.

2. Direct reversal of a lesion and is error-free

3. In E.coli, The response is adaptive because it is induced by low levels of alkylating agents and gives increased protection against the lethal and mutagenic effects of the high doses

Excision repair

1. Including

nucleotide excision repair (NER)

base excision repair (BER)

2. Ubiquitous mechanism repairing a variety of lesions.

3. Error-free repair

Nucleotide excision repair (NER)

1. An endonuclease

cleaves DNA a precise

number of bases on

both sides of the lesions

(e.g. in E.coli, UvrABC

Endonulcease removes

pyrimidine dimers)

2. Excised lesion-DNA

fragment is removed

3. The gap is filled by

DNA polymerase I

and sealed by ligase

Base excision repair (BER)

A specialized form of excision repair which

deals with any base mispairs produced

during replication and which have escaped

proofreading

Mismatch repair

The parental strand is methylated at N6 position of all As in GATC sites, but methylation of the daughter

strand lag a few minutes after replication

MutH/MutS recognize the mismatched

base pair and the nearby GATC

DNA helicase II, SSB, exonuclease Iremove the DNA fragment including the mismatch

DNA polymerase III & DNA ligase fill in the gap

Essay questions

1. How to explain the mechanisms of semi-conservative replication and semi-discontinuous replication? How to verify them by experiments?

2. How about the differences between prokaryotic and eukaryotic DNA replication?

3. How about the main types of DNA damage? and the main repair mechanisms?

DNA recombination

- Homologous recombination

- Site-specific recombination

- Transposition

An important reason for variable DNA sequencesamong different populations of the same species

Homologous recombination

1. Homologous duplicated chromosomes line up in parallel in metaphase I.

2. The nonsister chromatids exchange equivalent sections by crossing over.

- The exchange of homologous regions between two DNA molecules

In diploid eukaryotes, it commonly occurs during meiosis

Crossing over

Haploid prokaryotes recombination

- between the replicated portions of a partially

duplicated DNA

- between the chromosomal DNA and acquired

“foreign” DNA, like plasmids or phages

Occurs between the two homologous duplex

Nick formation

RecA-ssDNA filament

Recombination-based DNA repair

Site-specific recombination

1. Exchange of non-homologous but specific pieces of DNA

2. Mediated by proteins that recognize specific DNA sequences.

Bacteriophage λ insertion

1. λ -encoded integrase (Int): makes staggered cuts in

the specific sites

2. Int and IHF (integration host factor encoded by

bacteria): recombination and insertion

3. λ -encoded excisionase (XIS): excision of the

phage DNA

Antibody diversity

H and L are all encoded by three gene segments: V, D, J

V D J

Two heavy chains (L) 250 15 5

Two light chains (H) 250 4

Enormous number (>108) of different H and L gene sequences can be produced by such a recombination

Transposition

1. Requires no homology between sequences nor

site- specific

2. Relatively inefficient

3. Require Transposase encoded by the transposon

Transposons

E. coli

- IS elements/insertion sequence

1-2 kb, comprise a transposase gene flanked by a short inverted

terminal repeats

- Tn transposon series

carry transposition elements and β-lactamase

(penicillin resistance)

Eukaryotic transposons

many are retrotransposons

Yeast Ty element encodes protein similar to RT

(reverse transcriptase)