1. Andrew J. Pierce TOX 780 Single and Double-strand Break Repair TOX 780 Andrew Pierce Microbiology, Immunology and Molecular Genetics Toxicology University of Kentucky Part II: Homologous Recombination

2. Andrew J. Pierce TOX 780 Homologous Recombination: What is it? Single strand breaks and gaps are repaired using the unbroken DNA strand as a donor of missing sequence information Analogously, a double-strand break can repair missing sequence information by using an unbroken double-stranded template supplied exogenously Sources of repair templates include: sister chromatids in late S, G2 phases of the cell cycle the parental homolog in diploid organisms repeated sequence elements Using the sister as a template for repair (late S-phase) Intramolecular information donor Intermolecular information donor Homologous chromosome or ectopic sequence repeat

3. Andrew J. Pierce TOX 780 Homologous Recombination: How does it work? In all cases, "recombination" involves Watson-Crick base-pairing between DNA single strands that were NOT synthesized together through semi- conservative replication, i.e. between strands that have not been previously paired together. "Homologous" technically refers to sequences that shared an evolutionary relationship with each other (eg maternal and paternal versions of Chromosome 5), but now is used more generically to mean any two sequences which share a large degree of sequence identity with each other, whether that similarity arose through an evolutionary mechanism, through simple coincidence, or through deliberate design. For double-stranded DNA, the initial step in homologous recombination between two homologous single strands is necessarily the removal of their initial semi-conservative replication partner strands and the identification of sequence complementary between the recombining strands.

4. Andrew J. Pierce TOX 780 Homologous Recombination: How does it work? (continued) Removal of replicative pairing strand: exonuclease (not identified) strand displacement (Rad51, DNA polymerase) Identification of complementarity and subsequent base-pairing: Rad51, Rad52 Exonuclease (?) Strand invasion and displacement (Rad51) Synthesis and displacement (pol  ?) Broken end Homologous sequence Complementary sequence annealing (Rad52) Exonuclease (?) Two broken ends

5. Andrew J. Pierce TOX 780 Single Strand Annealing (SSA) Reaction required two broken ends with direct sequence repeats Involves obligate deletion of all sequences between the repeats Typically involves a double-strand break between formerly intramolecular repeats Activities required: Exonuclease to expose single strands (unidentified) Single-strand protection from degradation (RPA) Single-strand annealing (Rad52) Flap endonuclease (ERCC1 / XPF) Gap filling synthesis (pol  /  ?) Nick ligation (XRCC1/Dnl III ? Dnl I ?) See http://www.cellectis.com/dsbr.html for animations http://www.cellectis.com/dsbr.html

6. Andrew J. Pierce TOX 780 Synthesis-Dependent Strand Annealing (SDSA) Reaction between a broken chromosome and an intact homologous duplex as a donor of sequence information Replaces the lost information in the broken locus with a copy from the information donor (gene conversion) Thought to generally occur between sister chromatids in late-S or G2 phase Activities required: Exonuclease to expose single strands (Rad50/Mre11/Nbs ?) Single-strand protection from degradation (RPA) Single-strand invasion and D-loop formation (Rad51) DNA synthesis to extend D-loop (pol  /  ?) D-loop migration (unidentified) D-loop disassembly (BLM ?) Annealing (Rad52 ?) Gap filling synthesis (pol  /  ?) Nick ligation (XRCC1/Dnl III? Dnl I?) Mismatch repair

7. Andrew J. Pierce TOX 780 Gene Conversion with Possibility of Crossing-Over (classical double-strand break repair model) Same conditions required as for SDSA Can lead to exchange of flanking markers (crossing-over) Activities required: Exonuclease to expose single strands (Rad50/Mre11/Nbs ?) Single-strand protection from degradation (RPA) Single-strand invasion and D-loop formation (Rad51) DNA synthesis to extend D-loop (pol  /  ?) Holliday junction branch migration (BLM/TopIII ?) Holliday junction resolution (unidentified) Nick ligation (XRCC1/Dnl III? Dnl I?) Mismatch repair

8. Andrew J. Pierce TOX 780 Holliday Junction