Maintenance of genomes Copying the genome sequence (replication) Repairing damage to the genome sequence Rearranging genome sequences

Maintenance of genomes Copying the genome sequence (replication) Repairing damage to the genome sequence Rearranging genome sequences

Recombination = Insertion or exchange of DNA sequences between donor and acceptor DNA molecules

Recombination Homologous recombination Site-specific recombination Transposition Different sets of proteins are responsible for the different types of DNA exchange.

Occurs in DNA repair, in crossing over in meiosis, and in gene rearrangements.

Figure 17.1b Genomes 3 (© Garland Science 2007) e.g. insertion of phage genomes into bacterial genome sequences. Catalyzed by sequence-specific recombinases.

Figure 17.1c Genomes 3 (© Garland Science 2007) Catalyzed by enzymes encoded in transposon sequences.

Figure 17.1a Genomes 3 (© Garland Science 2007) Occurs in DNA repair, in crossing over in meiosis, and in gene rearrangements.

Homologous recombination Homologous recombination requires a free, non-protected end of double-stranded DNA at least two regions (> 100 bp) of homologous DNA, e.g. two copies of a chromosome

The RecBCD system of E. coli is the best understood system for homologous recombination RecC RecB RecD

A free, unprotected end of double-stranded DNA, e.g. a double strand break, is required for binding of RecBCD. RecBRecB RecDRecD C BD

Activity of the RecBCD complex Chi sites (5’-GCTGGTGG-3’) occur on average every 6 kb in the genome sequence of E. coli

3’ overhangs after processing by RecBCD 3’

RecA is a single-strand-binding protein that mediates base pairing of homologous DNA sequences ≈ one RecA molecule every 3 nucleotides

Formation of a heteroduplex and extension of DNA 3’ ends Strand invasion mediated by RecA 5’ 3’

After ligation of DNA strands: DNA heteroduplex with two Holliday junctions Holliday junctions

RuvA and RuvB proteins specifically bind to Holliday junctions and catalyze branch migration

RuvA/B Branch migration (DNA exchange) RuvA is a DNA-binding protein that recognizes Holliday junctions. RuvB is a hexameric helicase that breaks H-bonds.

RuvC is an endonuclease that cleaves DNA at Holliday junctions leaving ends that can be ligated by DNA ligases.

Figure 17.8 Genomes 3 (© Garland Science 2007)

Depending on the cuts made by RuvC, crossover or non- crossover products result

Homologous recombination and DNA repair

Insertion of a foreign gene into a genome by homologous recombination Foreign gene Free double- stranded end Homologous sequences Genome sequence Homologous recombination Genome sequence Foreign gene Free double- stranded end

In addition to the RecBCD system E. coli has a RecE and RecF system involved in repairing specific DNA damages. Components of the RecE and RecF systems can replace RecBCD. Similarly, RuvA, RuvB, and RuvC are not essential for homologous recombination in E. coli but can be substituted by proteins that have analogous functions. Proteins similar to E. coli RecABCD are present in eukaryotes.

RecA-like proteins Rad51 (humans) RadA (Archaeglobus fulgidus) RecA

Homologous recombination in meiosis recombination Cross-over products

Homologous recombination mediates pairing of homologous chromosomes in meiosis

Homologous recombination in meiosis Spo11 introduces double-strand breaks at many sites of chromatids (Complex of Mre11, Rad50, Xrs2)

Co-localization of Rad51 and Dmc1

Crossover or non- crossover products result Rad51C / XRCC3

Homologous recombination in meiosis recombination Cross-over products

A number of additional proteins are involved in homologous recombination. E.g. RecQ proteins promote noncrossover recombination.

In addition to the RecBCD system E. coli has a RecE and RecF system involved in repairing specific DNA damages. Components of the RecE and RecF systems can replace RecBCD. Similarly, RuvA, RuvB, and RuvC are not essential for homologous recombination in E. coli but can be substituted by proteins that have analogous functions. Proteins similar to E. coli RecABCD are present in eukaryotes.

Homologous recombination in meiosis Spo11 introduces double-strand breaks at many sites of chromatids (Complex of Mre11, Rad50, Xrs2)

Recombination frequencies are used to make genetic maps of chromosomes Physical (blue) and genetic (red) maps of a region of a yeast chromosome

Gene conversion is a special type of homologous recombination Mating type switching in yeast

Model for homologous recombination in mating type switching in yeast