Chapter 6 Molecular Biology of DNA Replication and Recombination Jones and Bartlett Publishers © 2005

DNA Replication problems Start (origin) of replication Initiation sites Elongation – direction is important Accuracy

How does DNA replicate? Possibilities: –Make two molecules, but with pieces mixed up. –Exact copy (conservative) –Use each strand to make copy (semi- conservative)

Replication of DNA by strand separation and copying of the two template strands using A-T and G-C base pairing (semi-conservative replication)

Experimental proof of semi-conservative replication of DNA Mehelson and Stahl experiment

Vicia faba Taylor, Woods and Hughes experiment Demonstrating semiconservative DNA replication

Use of a thymidine analog (BUdR) provides cytological proof that DNA in chromosomes also replicates semi-conservatively

Replication of a circular DNA molecule through a  structure ( in a plasmid)

Replication can be uni- or bi-directional During  -form replication, both parental DNA strands remain intact. DNA replication begins at special sequences called “origins of replication”. A single DNA molecule may have one (E. coli chromosome) or many origins (a human chromosome can have more than a thousand origins).

Replication of a circular DNA molecule by a “rolling circle” mechanism During rolling circle replication, one of the template DNA strands is cut to create a primer 3’-OH end.

For every round of replication, the tail of the rolling circle becomes one unit longer One strand of the rolling circle grows continuously while the other is made in small pieces (Okazaki fragments). This is common in phages.

Replication of a linear eukaryotic chromosome

Speed of replication E. coli and other prokaryotes have faster replication than eukaryotes: 1500 bp per second. (30 minutes, but 20 min. gen. time). Drosophila has multiple origins of replication, and can make 50 nucleotide pairs per second. Typical eukaryotes have origins every 40,000 bp, and can replicate all chromosomes in 5-10 hours.

First Enzyme Found to Be Involved in DNA Replication DNA polymerase III – covalent addition of nucleotides to the DNA chain. Requirements: deoxynucleoside triphosphates, Mg ++ ions, template DNA, and a primer

DNA Polymerase Characteristics Absolute requirement for a template strand, and new base selection is template driven. Polymerization is always in the 5’  3’ direction on the new strand, antiparallel to the template strand. Initiation of new chain growth is not possible without a primer with a 3’ OH

Other Enzymes DNA helicase – unwinds DNA DNA gyrase – relaxes supercoils –Also called topoisomerase II primase – synthesizes RNA primer, 5-12 bp long 5’  3’ exonuclease – removal of primer DNA ligase – joins ends of DNA

Model of an E. coli DNA replication fork showing the many proteins that play a role there

Prevention of knotting of DNA (as strands are separated) by DNA gyrase working ahead of the DNA replication fork

DNA Replication

The components that are different in RNA relative to DNA

Priming of DNA synthesis with an RNA segment primosome- polymerase alpha, other polypeptides make ~12 nucleotides of RNA, then ~23 DNA nucleotides.

New DNA chains are initiated by short RNA primers

Addition of a deoxynucleotide to the 3’-OH end of a primer chain

A misinserted deoxynucleotide is excised by the proofreading exonuclease function of DNA polymerase

One side of the fork grows continuously (leading side) while the other side grows by making small DNA pieces (lagging side)

Sequence of events in the joining of adjacent precursor fragments in eukaryotes

Accuracy of replication If human DNA error rates were one in a 10 5 base pairs, there would be 60,000 mistakes for each cell division cycle. The actual rate is 1 in 10 billion, or about one mistake for every three cell divisions. 3’ –to- 5’ exonuclease activity of DNA polymerase results in proofreading.