Chapter 5

•General Recombination

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)

Repair of replication forks

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

RecBCD/MRN

RecA

RuvA-RuvB

RuvC

DNA pol

Spo11

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

Triplex DNA formed by Triplex DNA formed by base “flipping”?base “flipping”?

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*†§ Rad51Rad51

How does a broken How does a broken strand find a strand find a homologous donor?homologous donor?

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, Rad50Xrs2 (Nbs1)

Rad51, Dmc1BRCA1, BRCA2

Spo11

DNA pol

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

DS break repair

Gene conversion

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

Gene conversion

Figure 5-65 Molecular Biology of the Cell (© Garland Science 2008)

Heteroduplex formation at sites of gene conversion and crossover

Figure 5-66 Molecular Biology of the Cell (© Garland Science 2008)

Gene conversion by mismatch correction

Resolution of recombinant intermediates in meiotic and mitotic cells

Resolution of recombination depends on where breaks occur

Patch Splice

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

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

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

Retrovirus lifecycle

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)

Structure of reverse transcriptase

Transposition of 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)

Transposition of non-retroviral like transposable elements

Figure 5-75 Molecular Biology of the Cell (© Garland Science 2008)

Expansion of repetitive elements in mouse and human lineages

Transposable elements near the -globin gene cluster

Alu - greenL1 - redBl - blueL1 - 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

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)

Inactivation of a marker gene by recombination

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