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

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

Recombination is similar to DNA hybridization

Resolution of recombination depends on where breaks occur Patch Splice

Spo11 RecBCD/MRN RecA DNA pol RuvA-RuvB RuvC

RecBCD Helicase/Nuclease Processes DS breaks to form ssDNA ends Loads RecA onto the ssDNA ends Destroys foreign DNA Binds ends and tracks along the DNA - ATP hydrolysis

The RecBCD complex prepares DNA ends for homologous recombination

Chi sites increase the rate of homologous recombination

The structure of the RecA/Rad51 filament

RecA/Rad51 filaments

RecA catalyzes synapse formation

How does a broken strand find a homologous donor? Rapid Exchange of A:T Base Pairs Is Essential for Recognition of DNA Homology by Human Rad51 Recombination Protein Molecular Cell, Vol. 4, 705–714, November, 1999, Ravindra C. Gupta,* Ewa Folta-Stogniew,† Shawn O’Malley,* Masayuki Takahashi,‡ and Charles M. Radding*†§ Rad51 Triplex DNA formed by base “flipping”?

RecA contains two DNA binding sites

RecA 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 RuvA: Holiday junction binding protein (tetramer) RuvB: ATP dependent helicase (hexamer)

An alternate representation of RuvAB

RuvC resolves Holiday structures

RecBCD RecA RuvA-RuvB RuvC MRX complex Mre11, Rad51, Dmc1 BRCA1, BRCA2 Xrs2 (Nbs1) Rad51, Dmc1 BRCA1, BRCA2 Spo11 DNA pol

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 Figure 5-67 Molecular Biology of the Cell (© Garland Science 2008)

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 5-72a Molecular Biology of the Cell (© Garland Science 2008)

Structure of reverse transcriptase Figure 5-72b 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 L1 - red Bl - blue L1 - 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 attP attB Integration Host Factor (IHF) attL attR

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

Figure 5-79b 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