Chapter 9

DNA Replication

How is DNA synthesized?

Parental strand is used as a template for the newly replicated strand

3 possible models for DNA replication

1. Semiconservative

2.
3.

Conservative
Dispersive

Experimental evidence for Semiconservative Model

Meselson and Stahl experiment (1958): 1. Incorporate heavy isotope of nitrogen (15N) into both strands of E. coli DNA, then allow replication in medium containing only the light isotope (14N) 2. Separate newly replicated DNA using density centrifugation

Predicted results for the 3 models of DNA replication

(conservative model disproved)

(dispersive model disproved)

DNA replication in eukaryotes is also semiconservative

Herbert Taylors experiment (1958):

http://mol-biol4masters.masters.grkraj.org/html/Prokaryotic_DNA_Replication1-Introduction.htm

Replicating Chromosome in E. coli

John Cairns (1963):

The E. coli chromosome is circular Preserves the integrity of the circular chromosome DNA replication initiates at a single point and proceeds from one or two replication forks

DNA Replication in E. coli starts at an origin and is bidirectional

DNA replication in eukaryotes starts at multiple origins and is bidirectional

Replication bubbles form at multiple origins on the linear eukaryotic chromosome

replicon: DNA replicated from a single origin

Drosophila chromosomes

DNA Synthesis
Requires DNA Polymerase 1. 3 DNA polymerases : I, II, and III - polymerase III : replicates most of the DNA 2. Synthesizes DNA in a 5 to 3 direction

3. Synthesizes DNA antiparallel to the parental strand

Nucleotide Addition : 5 to 3 Synthesis

5 5

Catalyzes formation of phosphodiester bond between 5 PO4 of deoxyribonucleotide and 3 OH on DNA strand - substrate is deoxynucleotide triphosphate

http://en.wikibooks.org/wiki/Medical_Physiology/Cellular_Physiology/DNA_and_Reproduction

Energy for polymerization comes from cleaving 2 phosphates from deoxyribonucleotide triphosphate

Nucleotide Addition : 5 to 3 Synthesis

Leading strand is synthesized continuously in direction of the movement of the replication fork DNA pol III : high processivity (synthesizes very long strand)

leading strand

lagging strand

Lagging strand is synthesized discontinuously (in pieces: Okazaki fragments) in direction opposite of movement of replication fork

Leading and Lagging Strand Synthesis requires primers

Primers are short sequences of nucleotides (RNA or DNA) which are base paired to the template DNA Primers provide a de novo 3OH end for an incoming deoxyribonucleotide

Leading and Lagging Strand Synthesis at the Replication Fork

(~150bp in eukaryotes)

Lagging Strand Synthesis

Four steps: 1. Primer synthesis 2. Elongation 3. Primer removal with gap filling 4. Ligation

Primer Synthesis
Primase synthesizes a primer (10-12 nucleotides long) complementary to DNA template strand

Elongation
DNA polymerase III adds deoxyribonucleotides to the 3 OH end of the primer

E. coli : 400 nt per second !

Proofreading activity of DNA polymerase

3 to 5 exonuclease activity removes mismatched base pairs - nuclease : degrades DNA - exonuclease cleaves from end - endonuclease cleaves phosphodiester bond between nucleotides
* every polymerase has 3 5 exonuclease activity ** only DNA pol I has 5 3 exonuclease activity

Elongation

Fidelity : a measure of polymerase accuracy at incorporating correct deoxynucleotide (E. coli : 1 mismatch in 109 base pairs !)

Primer Removal and Gap Filling

why is primer made of RNA ? - primers are not always exact matches to template RNA primer can be removed and correct DNA sequence added by pol III

53 exonuclease activity of DNA polymerase I removes primer 53 polymerase activity of DNA polymerase I fills in the gap with DNA

Ligation
DNA Ligase seals up nicks left after DNA polymerase has filled in the gaps
ligase : catalyzes formation of a phosphodoester bond from a 5 phosphate and 3 OH

DNA synthesis begins at an origin

Step 1: Initiator proteins (dna A) bind origin (ori C) ori C : specific DNA sequence, ~245 bp long

Binding of initiator proteins at origin denatures DNA

Helicase Unwinds DNA, Primase Synthesizes a Primer

Helicase + Primase = Primosome

DNA Polymerase Synthesizes the Leading Strand

Primosomes generate more primers for lagging strand synthesis

Continuous and discontinuous DNA synthesis occur at replication fork

continuous synthesis

discontinuous synthesis

At replication fork : primosome + 2 DNA pol IIIs + ssb proteins

ssbs bind to single-stranded DNA and keep it from reannealing during replication

replisome : primosome + 2 DNA pol IIIs - move as a unit along DNA

holoenzyme : protein complex with all associated subunits DNA pol III : 10 subunits total - 3 form core enzyme required for activity - subunit : clamp that holds polymerase onto DNA processivity

for overall movement in direction of replication fork : lagging strand must loop through DNA pol III to allow 53 synthesis of Okazaki fragment away from replication fork

polymerase cycling : moving off and on the lagging strand template as Okazaki fragments are completed

Events at the replication fork during polymerase recycling

2. release of primase

3. recruit

clamp

1. synthesis of RNA primer

5. reattach primase further along lagging strand

4. recruit DNA pol III, Okazaki fragment synthesis 5 3

Supercoiling
Topoisomerases can put in or remove supercoils from the DNA

linkage number (L) : number of turns of 1 helix around the other

negative : circular DNA winds around itself in the opposite direction as the twist of helix (left handed)

positive : circular DNA winds around itself in the same direction as the twist of helix (right handed)

Topoisomerases
Change the supercoiling of the DNA by increasing or decreasing the linkage number Type I Topoisomerases cut one of the DNA strands, insert unbroken end in opening Type II Topoisomerases cut both DNA strands, insert unbroken double helix through opening i.e., gyrase

Topoisomerases
Required for DNA replication As helix opens during DNA replication, positive supercoils occur in front of the replication fork gyrase removes those supercoils so that DNA replication can proceed

Topoisomerase

Termination of Replication in E. coli

ter sites (directly opposite oriC site) bound by termination protein (encoded by tus gene : termination utilization substance)

Intertwined chromosomes have to be separated by topoisomerase (type II)

Other models to replicate circular chromosomes

1. Rolling Circle

2. D loop

Rolling Circle Replication in E. coli Plasmids

D-Loop Replication in Mitochondria and Chloroplasts

origins are at different places on parental template strand (Displacement loop)

unidirectional leading-strand synthesis from both strands

Rates of DNA Synthesis during replication

E.coli : 25,000 bp/min

eukaryotic : 2000 bp/min

Eukaryotic DNA Replication

differences from prokaryotes : 9 different DNA polymerases - : major one in replication - : makes Okazaki fragment primers RNA primers removed by RNAse, not DNA pol Multiple origins - yeast : ARS (autonomously replicating sequences) Telomeres: special sequences at chromosome ends similarities to prokaryotes : specific sequences at origins are bound by proteins - called Origin Replication Complex (ORC) in eukaryotes all of the enzymatic processes

How Are the Ends of Chromosomes Preserved during DNA Replication?

Telomerase
RNA + enzyme complex that binds and extends 3 end of linear chromosomes ** ultimate goal : not to increase length, but preserve telomere 1. Telomerase RNA base pairs with single-stranded 3 end of DNA

several times

2.

Telomerase extends telomere by reverse transcription (RNA from telomerase is template) adds GGGGTT (or similar) sequence Telomerase translocates to extended 3 end

3.

- then primase, polymerase, and ligase make DNA strand complementary to the new telomere sequence

Telomerase
1. Telomerase RNA base pairs with DNA

Telomerase
2. Telomere extension occurs reverse transcription

Telomerase
3. Telomerase translocates to extended 3 end

Telomerase
4. Telomerase extends 3 end of telomere reverse transcription

How Telomerase Extends the 3 End of a Linear Chromosome

can be <150 bp up to 10-20 kb in length

exact size of telomere may fluctuate from one round of DNA replication to the next, but . . . ** genomic information adjacent to telomere is preserved **

Telomere binding proteins regulate telomeres

TRF1 : # of proteins bound to telomeres determines whether telomerase should extend telomeres TRF2 : prevent end-end fusion of different chromosomes

van Steensel et al., Cell 92::Pages 401413 (1998)

DNA packaging and replication model