Homologous Recombination Strand Invasion I. Homologous Recombination a. Genetic exchange between a pair of homologous chromosomes b.

Repairs double-stranded breaks (both sides of DNA); rearranges DNA sequences; facilitates chromosome segregation Basic Steps a. Double-stranded break occurs (radiation or Spo11 protein) b. Resection: 5 DNA removed by MRN complex i. MRN complex (MRX complex in yeast): binds to double-strand breaks; tethers broken ends; initiates resection before homologous recombination ii. Activates checkpoint kinase ATM in response to DNA damage Strand Invasion a. Pairing of single-stranded broken DNA with complementary double-stranded unbroken DNA b. Recipient double-stranded DNA can be identical sister chromatids (common) or similar but non-identical homologous chromosomes (meiosis) c. Displacement loop (D-loop): forms between invading 3 end of ssDNA and recipient dsDNA Remaining Steps a. DNA polymerase extends end of invading ssDNA b. Invading single-stranded DNA rejoins, forming 2 Holliday Junctions c. Branch migration: Holliday junctions move away from site of formation, switching out DNA Products a. 2 horizontal cuts = non-crossover products b. 1 vertical & 1 horizontal = crossover products

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RecA & Rad51 Proteins I. II. Intro a. RecA is found in E. coli; Rad51 is its homolog, present in most all eukaryotes Function in HR a. A nick in dsDNA enables strand invasion by ssDNA; heteroduplex is formed b. RecA catalyzes base pairing of ssDNA and complimentary dsDNA c. RecA can hold a single-strand and double-strand molecule at the same time (multiple DNA binding sites) d. First, a non-base-paired complex is formed that converts to a three-stranded structure as soon as homologous DNA is formed i. Presumably unstable due to its unusual form of DNA e. Spins out heteroduplex and a displaced single-strand PALB2 & BRCA2 a. Rad51 interacts with PALB2/BRCA2 b. BRCA2 regulates intracellular localization and DNA-binding ability of Rad51 c. BRCA2 inactivation leads to genomic instability and tumorigenesis

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Spo11 I. Spo11: Intro a. S. cerevisae: meiotic recombination results from the formation and repair of double-strand breaks b. Spo11 was one of the first meiotic recombination genes to be identified

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c. Type II topoisomerase that initiates homologous recombination by making targeted double-strand breaks in DNA d. Based on phylogenetic trees, the version of Spo11 in eukaryotes emerged in the last common ancestor of eukaryotes and archaea Overview a. Spo11 breaks DNA (topoisomerase-like reaction), generating a covalent protein-DNA intermediate i. Tyrosine at the active site of a dimer of Spo11 involved b. Spo11 is removed by an endonucleolytic cleavage c. Spo11 covalently attaches to short oligonucleotide (tags that mark where Spo11 made a double-strand break) d. Spo11 oligonucleotide mapping can be used to study changes in double-strand break patterns; methodology can be extended to mice, fission yeast, and other organisms Planned a. CDK (cyclin-dependent kinases) promote double-strand breaks in yeast b. CDK phosphorylates Mer2 (one of 9 yeast proteins required with Spo11 for double-strand breaks) c. ATM (involved in ataxia telangiectasia huh, imagine that) controls meiotic double-strand break numbers d. ATM is necessary to govern a negative feedback loop that restrains Spo11 and therefore limits DSBs.

Branch Migration I. II. Homologous Recombination a. Can only take place between DNA that have large regions of similar DNA sequences Branch Migration a. The branch point (Holliday junction) moves across the DNA, displacing a region of the dsDNA molecule Spontaneous Branch Migration a. Back-and-forth process that is not enzyme driven b. Proceeds equally in either direction; little overall progress is made c. No heteroduplex DNA (DNA that consists of strands from two different DNA molecules) is produced as a result Protein-directed Branch Migration a. Energy-dependent process that is driven by specialized DNA helicases b. Proceeds in one direction c. A region of heteroduplex DNA that is thousands of base pairs is produced as a result

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Holliday Junctions I. Simple Holliday Junction a. RuvA & RuvB bind to 4 strand DNA structure and migrate the strands through each other b. The binding of RuvC to RuvAB is thought to cleave the DNA strands and resolve the Holliday junction c. Vertical cut: crossover product; Horizontal cut: non-crossover product Complicated Holliday Junction (Double Holliday Junction) a. Two vertical or horizontal cuts: non-crossover resolution b. One horizontal and one vertical: crossover products c. Can cause gene conversion, divergence from the expected distribution of alleles

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