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

A misinserted deoxynucleotide is excised by the proofreading exonuclease function of DNA polymerase Q1. Why is a mismatch a problem? Q2. What is the consequence if proofreading doesn’t catch a mistake?

Addition of a dideoxynucleotide to the 3’-OH end of a DNA chain terminates chain elongation

The chain termination method of DNA sequencing

DNA sequencing machines use fluorescent dideoxynucleotides

A “shotgun sequencing” approach for sequencing of a large DNA molecule

Analogs of normal deoxynucleotides useful as anti-AIDS agents Dideoxynucleosides are used because they can cross cell membranes more easily than can dideoxynucleotides

Excision repair of a mismatched base pair Mismatch repair can result in ‘gene conversion’.

Variations from normal 2:2 segregation (gene conversion) can result from mismatch repair

Molecular details of recombination How are heteroduplexes formed as intermediate steps in recombination? First model that described molecular recombination was suggested by Holliday in Holliday junction

Symmetric model of homologous recombination showing some of the intermediate steps and structures Note the 2 nicks in the same location in the two duplexes in step (B). This is followed by strand exchange and Holliday junction formation (C)

An asymmetric model of homologous recombination Recombination is initiated by a cut in only one of the 2 recombining duplexes (A) followed by strand invasion (B) and Holliday junction formation (C)

A recombining molecule showing the parental and the recombinant duplexes joined at the Holliday junction

Enzymes involved in recombination in E. coli Obvious ones: exonuclease, ligase Others: RecA RecBCD RuvC – Holliday junction-resolving enzyme

Resolution of a Holliday junction can occur by 2 alternative cleavage pathways

3D view of a Holliday junction

Double-strand break & repair The current favored model for homologous recombination is the double-strand break and repair model. A broken duplex interacts with an intact duplex. Gaps and sticky ends on broken duplex invade intact duplex. Displacement results (D loop).

Model for the recombinational repair of a double strand break in DNA