Chapter 5 General Recombination Repair of replication forks

Chapter 5 • General Recombination

Repair of replication forks Figure 5 -53 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

Repair of replication forks Figure 5 -53 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)

General recombination transfers information from one DNA strand to another

DNA crossovers create heteroduplex DNA

General recombination in meiosis

ds break General recombination in meiosis synapse strand invasion heteroduplex formation branch migration resolution

Recombination is similar to DNA hybridization

Resolution of recombination depends on where breaks occur Patch Splice

Spo 11 Rec. BCD/MRN Rec. A DNA pol Ruv. A-Ruv. B Ruv. C

Rec. BCD Helicase/Nuclease Processes DS breaks to form ss. DNA ends Loads Rec. A onto the ss. DNA ends Destroys foreign DNA Binds ends and tracks along the DNA - ATP hydrolysis

The Rec. BCD complex prepares DNA ends for homologous recombination

Chi sites increase the rate of homologous recombination

The structure of the Rec. A/Rad 51 filament

Rec. A/Rad 51 filaments

Rec. A catalyzes synapse formation

How does a broken strand find a homologous donor? Triplex DNA formed by base “flipping”? Rapid Exchange of A: T Base Pairs Is Essential for Recognition of DNA Homology by Human Rad 51 Recombination Protein Ravindra C. Gupta, * Ewa Folta-Stogniew, † Shawn O’Malley, * Masayuki Takahashi, ‡ and Charles M. Radding*†§ Molecular Cell, Vol. 4, 705– 714, November, 1999, Rad 51

Rec. A contains two DNA binding sites

Rec. A catalyzes branch migration

Figure 5 -58 Molecular Biology of the Cell (© Garland Science 2008)

The Holliday junction

A EM micrograph of a Holliday junction

Ruv proteins catalyze double branch migration Ruv. A: Holiday junction binding protein (tetramer) Ruv. B: ATP dependent helicase (hexamer)

An alternate representation of Ruv. AB

Ruv. C resolves Holiday structures

Spo 11 Rec. BCD MRX complex Mre 11, Rad 50 Xrs 2 (Nbs 1) Rec. A DNA pol Ruv. A-Ruv. B Ruv. C Rad 51, Dmc 1 BRCA 1, BRCA 2

DS break repair Figure 5 -59 Molecular Biology of the Cell (© Garland Science 2008)

Gene conversion

Gene conversion Figure 5 -63 Molecular Biology of the Cell (© Garland Science 2008)

Heteroduplex formation at sites of gene conversion and crossover Figure 5 -65 Molecular Biology of the Cell (© Garland Science 2008)

Gene conversion by mismatch correction Figure 5 -66 Molecular Biology of the Cell (© Garland Science 2008)

Resolution of recombinant intermediates in meiotic and mitotic cells

Resolution of recombination depends on where breaks occur Patch Splice

Mismatch detection prevents recombination of similar sequences

Recombination controls yeast mating types

Chapter 5 • Site-Specific Recombination

The human genome contains many transposable elements

Table 5 -3 Molecular Biology of the Cell (© Garland Science 2008)

Bacterial transposable elements

Cut-and-paste transposition

The structure of a transposase bound to DNA

Replicative cut-and-paste transposition

Retrovirus lifecycle Figure 5 -71 Molecular Biology of the Cell (© Garland Science 2008)

Structure of reverse transcriptase Figure 5 -72 a Molecular Biology of the Cell (© Garland Science 2008)

Structure of reverse transcriptase Figure 5 -72 b Molecular Biology of the Cell (© Garland Science 2008)

Transposition of retroviral like transposable elements

Transposition of non-retroviral like transposable elements Figure 5 -74 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

Transposition of non-retroviral like transposable elements Figure 5 -74 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)

Expansion of repetitive elements in mouse and human lineages Figure 5 -75 Molecular Biology of the Cell (© Garland Science 2008)

Transposable elements near the -globin gene cluster Alu - green L 1 - red Bl - blue L 1 - yellow

The human genome contains many transposable elements

Table 5 -3 Molecular Biology of the Cell (© Garland Science 2008)

Conservative site-specific recombination can rearrange DNA

Insertion of lambda DNA into a bacterial chromosome

Insertion of lambda DNA into a bacterial chromosome att. P att. B Integration Host Factor (IHF) att. L att. R

The lambda phage life cycle

Use of site-specific recombination to control gene expression

Inactivation of a marker gene by recombination

Inactivation of a marker gene by recombination Figure 5 -79 Molecular Biology of the Cell (© Garland Science 2008)

Inactivation of a marker gene by recombination Figure 5 -79 a Molecular Biology of the Cell (© Garland Science 2008)

Figure 5 -79 b Molecular Biology of the Cell (© Garland Science 2008)

Points to understand: The differences between site-specific and general recombination The consequences of each type of recombination The three types of transposable elements How the elements move How the TEs relate to viruses and phage Conservative site specific recombination and how it is used by cells and experimental biologists