Mechanism of Homologous Recombination (HR) Isaac Manuel Torres Bermeo Ghadir Sindi Prateek Kulkarni

Homologous recombination Is the exchange of genetic information between allelic sequences. Is a mechanism for the repair of damaged chromosomes.

Characteristics Maintains genome integrity during both meiosis and mitosis. Ensures genome stability. Essential for generating genetic diversity. Serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes.

Exchange of nucleotide sequences between two similar or identical DNA molecules. In mitosis: repair double- stand breaks (DSB) –DNA repair In meiosis: provide new combinations of DNA sequences –DNA mutation https://en.wikipedia.org/wiki/Homologous_recombination#/media/File:HR_in_meiosis.svg

Biological roles of HR Sung and Klein, 2006

Mechanisms of HR The actual mechanism can be divided separately into three steps: Presynapsis Synapsis Postsynapsis

Initiation of HR by DSB Processing of the DSB by nucleolytic resection to give single-strand tails with 3’-OH ends. San Filippo et al., 2008

Formation of Recombinase filament on the ssDNA ends Sung and Klein, 2006

Strand invasion into homologous sequence Formation of D-loop intermediate. DNA polymerase extension from 3’ end. Lodish et al., Molecular Cell Biology, 5th ed. Fig 23-31

Formation of two crossed strand or Holliday junction (HJ) Resolution of the HJs to give crossover or noncrossover products. San Filippo et al., 2008

Holliday Junction

Synthesis-Dependent Strand Annealing (SDSA) San Filippo et al., 2008

Single-Strand Annealing (SSA) https://openi.nlm.nih.gov/detailedresult.php?img=PMC4224172_795fig3&req=4

HR Factors Mediates formation of DNA joints that links homologous DNA molecules. Recombinases BRCA2: interact with Rad51 and forms a complex RPA (Replication Protein A): Nuclear protein that binds to ssDNA with high affinity. Has stimulatory/inhibitory effect on the assembly of presynaptic filament. Rad51 and others Rad51 somatic & meiotic cells Dmc1  meiotic cells

Action of recombination mediators RPA (Replication Protein A) has higher ssDNA binding affinity than Rad51 Several recombination mediators like BRCA and Brh2 helps RAD51 to bind ssDNA and alleviate the inhibitory effect of RPA Sung and Klein, 2006

Individual study of mediators San Filippo et al., 2008

Regulation of HR by DNA helicases Inappropriate or untimely HR can result in deleterious consequenses. Formation of crossovers during HR can lead to loss of heterozygosity. If LOH event involve regulatory genes of cell growth and differentiation can lead to cancer. HR can interfere with post replication repair result in DNA damage checkpoint mediated cell cycle arrest.

Regulation of HR by Srs2 helicases Srs2 helicase dismantle the Rad51 presynaptic filament in 3’ to 5’ direction The bare ssDNA becomes available for RPA to bind to prevent reloading of Rad51. This results in unwanted HR events. Sung and Klein, 2006

Regulation of HR by BLM helicases This is specifically where two Holliday Junctions occur. The two HJs are pushed inwards by DNA helicases to form a hemicatenane. This is further dissolved by a topoisomerase to give non cross-over recombinants. This mechanism prevents events like loss of heterozygosity and chromosome rearrangements Sung and Klein, 2006

HR Application Knockout Mouse: It is a type of mouse where both the alleles of a particular gens are replaced by inactive alleles. This is accomplished by HR. bio.davidson.edu; cellmigration.org

CRISPER-Cas9 Precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway. New England Biolabs

References Jeremy M. Berg (2007) Biochemistry 6th, W.H. Freeman and Company. Lodish et al., (2007) Molecular Cell Biology, 6th, W.H. Freeman and Company Patrick Sung and Hannah Klein (2006). Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nature Reviews Mol.Cell Biol. 7, 739- 750. San Filippo, J., Sung, P., & Klein, H. (2008). Mechanism of Eukaryotic Homologous Recombination. Annual Review of Biochemistry, 77(1), 229–257.