DNA Replication in Prokaryotes and Eukaryotes

1. Overall mechanism

2. Roles of Polymerases & other proteins

3. More mechanism: Initiation and Termination

4. Mitochondrial DNA replication

DNA replication is semi-conservative, i.e., each daughter duplex molecule contains one new strand and one old.

Does DNA replication begin at

the same site in every replication

cycle?

Electron microscope image of an E. coli chromosome being

replicated.

Structure (theta, θ) suggests replication

started in only one place on this chromosome. Fig. 20.9

Does DNA replication begin at the same site in every replication cycle?

Experiment:1. Pulse-label a synchronized cell population

during successive rounds of DNA replication with two different isotopes, one that changes the density of newly synthesized DNA (15N), and one that makes it radioactive (32P).

2. DNA is then isolated, sheared, and separated by CsCl density gradient ultra-centrifugation.

3. Radioactivity (32P) in the DNAs of different densities is counted.

1st

Prior to 1st replication cycle, 15N (which incorporates into the bases of DNA) was added for a brief period

Prior to 2nd replication cycle, cells were pulsed with 32P (which gets incorporated into the phosphates of replicating DNA)

15N - heavy isotope of Nitrogen32P - radioactive isotope of phosphorus

DNA is isolated, sheared into fragments, and separated by CsCl-density gradient centrifugation.

Blow up of the last 2 rows of DNA in the previous slide (i.e., labeled DNA, and labeled, sheared DNA).

Labeled DNALabeled, sheared DNA

Same Origin

Random Origins

Conclusion:Replication of bacterial chromosome starts at the same place every time

Result: ~50% (the most possible) of the incorporated 32P was in the same DNA that was shifted by 15N

Using Electron Microscopy (EM) to Demonstrate that DNA Replication is

Bi-Directional

- Pulse-label with radioactive precursor (3H-thymidine)

- Then do EM and autoradiography.

- Has been done with prokaryotes and eukaryotes.

Conclusion: eukaryotic origins also replicate bi-directionally!

Drosophila cells were labeled with a pulse of highly radioactive precursor, followed by a pulse of lower radioactive precursor; then replication bubbles were viewed by EM and autoradiography.

Fig. 20.12 in Weaver

Another way to see that DNA replication is Bi-directional --

Cleave replicatingSV40 viral DNA with a restriction enzyme thatcuts it once.

Similar to Fig. 21.2 in Weaver 4

Organism # of replicons Averagelength ofreplicon

Velocity offorkmovement

Escherichia coli (bacteria) 1 4200 kb 50,000bp/min

Saccharomyces cerevisiae(yeast)

500 40 kb 3,600 bp/min

Drosophila melanogaster(fruit fly)

3,500 40 kb 2,600 bp/min

Xenopus laevis (frog) 15,000 200 kb 500 bp/minMus musculus (mouse) 25,000 150 kb 2,200 bp

/minHomo sapiens 10,000 to

100,000Š 300 kb

Replicon - DNA replicated from a single origin

Eukaryotes have many replication origins.

Enzymology of DNA replication: implications for mechanism

1. DNA-dependent DNA polymerases

– synthesize DNA from dNTPs

– require a template strand and a primer strand with a 3’-OH end

– all synthesize from 5’ to 3’ (add nt to 3’ end only)

Movie – DNA polymerization

Note: what happens to the P-P?

Comparison of E.coli DNA Polymerases I and III

1 subunit

10 subunits

Proofreading Activity

Insertion of the wrong nucleotide causes the DNA polymerase to stall, and then the 3’-to-5’ exonuclease activity removes the mispaired A nt. The polymerase then continues adding nts to the primer.

Fig. 20.15 in Weaver 4

If DNA polymerases only synthesize 5’ to 3’, how does the replication fork move directionally?

• Lagging strand synthesized as small (~100-1000 bp) fragments - “Okazaki fragments” .

• Okazaki fragments begin as very short 6-15 nt RNA primers synthesized by primase.

2. Primase - RNA polymerase that synthesizes the RNA primers (11-12 nt that start with pppAG) for both lagging and leading strand synthesis

Pol III extends the RNA primers until the 3’ end of an Okazaki fragment reaches the 5’ end of a downstream Okazaki fragment.

Lagging strand synthesis (continued)

Then, Pol I degrades the RNA part with its 5’-3’ exonuclease activity, and replaces it with DNA. Pol I is not highly processive, so stops before going far.

At this stage, Lagging strand is a series of DNA fragments (without gaps).

Fragments stitched together covalently by DNA Ligase.

3. DNA Ligase - joins the 5’ phosphate of one DNA molecule to the 3’ OH of another, using energy in the form of NAD (prokaryotes) or ATP (eukaryotes). It prefers substrates that are double-stranded, with only one strand needing ligation, and lacking gaps.

Ligase will join these two G--G--A--T--C--C--T--T--G--A--T--C--C| | | | | | | | | | | | |C--C--T--A--G G--A--A--C--T--A--G--G

Ligase will NOT join thesetwo.

G--G--A--T--C--C--T--T--G--A--T--C--C| | | | | | | | | | | |C--C--T--A--G C--A--A--C--T--A--G--G

Ligase will NOT join thesetwo.

G--G--A--T--C--C--T--T--G--A--T--C--C| | | | | | | | | | | |C--C--T--A--A G--A--A--C--T--A--G--G

Ligase will NOT join thesetwo.

G--G--A--T--C--C--T--T--G--A--T--C--C| | | | | | | | | | | |C--C--T--A--G G--T--A--C--T--A--G--G

Ligase will NOT join thesetwo. C--C--T--A--G C--T--A--C--T--A--G--G

DNA Ligase Substrate Specificity

2

1

+ AMP3'

PAMP

P

AMP+

HO

3'P

5'

Ligase

NAD

1 2

1

3'NMN

HOP

3'5'

P

Ligase

NAD NMN+AMP

Mechanism of Prokaryotic DNA Ligase

Ligase cleaves NAD and attaches to AMP.

Ligase-AMP binds and attaches to 5’ end of DNA #1 via the AMP.

The 3’OH of DNA #2 reacts with the phosphodiester shown, displacing the AMP-ligase.

AMP & ligase separate.

(Euk. DNA ligase uses ATP as AMP donor)

Movie - Bidirectional Replication: Leading and lagging strand synthesis

Replisome - DNA and protein machinery at a replication fork.

Other proteins needed for DNA replication:

4. DNA Helicase (dnaB gene) – hexameric protein, unwinds DNA strands, uses ATP.

5. SSB – single-strand DNA binding protein, prevents strands from re-annealing and from being degraded, stimulates DNA Pol III.

6. Gyrase – a.k.a. Topoisomerase II, keeps DNA ahead of fork from over winding (i.e.,

relieves torsional strain).

DNA Helicase (dnaB gene) Assay

Fig. 20.21 in Weaver

Helicase – the movie

Replication Causes DNA to Supercoil

Rubber Band Model of Supercoiling DNA

DNA Gyrase relaxes positive supercoils by breaking and rejoining both DNA strands.