Homologous Recombination Hr Proteins

 Creative BioMart Homologous Recombination Hr Proteins Product List
 Homologous Recombination Hr Proteins Background

Studies in yeast have shown that in somatic cells there is a bias toward recombination using the sister chromatid rather than the homologous chromosome, though there is evidence of homologous recombination (HR) between homologs in G1. Cohesion of sister chromatid ensures proximity of the recombination template and stabilizes interactions between them. HR repairs DSBs and replication associated breaks in the S and G2 phases of the cell cycle; ssDNA nicks and gaps can also be repaired by HR. Homologous recombination in meiosis involves similar mechanisms to mitotic HR and occurs after Spo11 initiates DSBs. Though functional HR promotes genome stability, it can also cause genomic instability when it acts inappropriately or in an unregulated manner. Proteins recruited to repair DSBs can be visualized by the formation of repair foci at lesions.

Mutations in genes involved in DNA repair have been implicated in human disease. For example, BRCA1 and BRCA2 are important factors in the homologous recombination pathway, and mutations in these proteins are associated with increased risk of cancer. Hereditary breast cancer from heterozygous germline BRCA mutations accounts for 5-7% of all cases of breast cancer, and patients with BRCA mutations have 50-80% risk of breast cancer and 30-50% risk for ovarian cancer, as well as a slightly increased risk for prostate and pancreatic cancers. It is generally thought that BRCA tumors have loss of heterozygosity (LOH) or reduction in BRCA expression, but LOH does not always happen in tumors and it is possible there is haploinsufficiency. Biallelic BRCA2, BRCA1, and PALB2 mutations are rare and cause Fanconi Anemia, which is associated with defects in interstrand crosslink (ICL) repair. Other diseases caused by HR defects include Nijmegen breakage syndrome (due to NBS1 mutation), Fanconi Anemia (ICL repair protein mutations), Bloom’s syndrome (BLM mutation), ataxia telangiectasia (ATM mutation), ataxia telangiectasia-like disorder (Mre11 mutations), Seckel syndrome (ATR and CtIP mutation), Werner syndrome (WRN mutation), and Rothmund-Thomson Syndrome (RECQL4 mutation). Thus, HR is important to DNA repair function.

Initiation of homologous recombination involves nucleolytic resection of the 5’ end at DSB sites to generate 3’ ssDNA overhangs. Mammalian DSB resection and its regulation in vivo are not well understand, but it is believed to be an important mechanism in DSB repair pathway choice, as it inhibits NHEJ and promotes HR. Other DSB repair pathways require resection as well: SSA begins similarly to HR, with fairly extensive resection, and MMEJ (or alt-NHEJ) also requires limited resection.

Resection is initiated by the nucleases MRN and CtIP. The MRN complex, composed of MRE11, RAD50, and NBS1, recognizes and binds DSB ends. RAD50 and MRE11 stabilize the break and tether the DNA ends, while NBS1 interacts with the ATM kinase, which phosphorylates and regulates many DNA repair proteins. MRE11 has ssDNA endonuclease, and 3’-5’ exonuclease activities (though 5’-3’resection occurs in vivo); its endonuclease activity is believed to be important in resection. MRN interacts with CtIP, which promotes resection. CtIP has a 5’ flap endonuclease activity independent of MRN, and its phosphorylation at numerous sites in response to DNA damage by cyclin-dependent kinase (CDK), ATM, and ATR, is believed to regulate its function. HR at complex breaks, like those with topoisomerase adducts or generated by IR, requires CtIP nuclease activity, while this nuclease activity is dispensable for CtIP’s role in HR repair of endonuclease breaks. CtIP has also been shown to enhance the nuclease activity of MRE11 in vitro, and is important for ssDNA formation in vivo. It has been suggested that MRN and CtIP may clip Ku from DNA ends similar to way they clip Spo11 off in meiosis.

A generally accepted model of resection is that MRN and CtIP initiate end resection by endonucleolytic cleavage of 5’ ends internal to break ends, releasing oligonucleotides. Biochemical studies with human proteins and studies in yeast suggest that there are two routes for more extensive resection, stimulated by RPA and MRN, and requiring either the DNA2 nuclease in complex with BLM-TOPIIIα-RMI1 or the EXO1 nuclease. Resection varies in length from a few hundred nucleotides to tens of kilobases, depending on the availability and location of homologous template.

After resection, the heterotrimeric ssDNA binding complex RPA binds the 3’ ssDNA overhangs. For HR to proceed, RPA needs to be exchanged for filaments of the RAD51 recombinase, which performs the essential homology search and strand invasion steps of HR. The RAD51-ssDNA nucleprotein filament is called the presynaptic filament. When the 3’ ssDNA invades a DNA duplex, it base pairs to a complementary strand and displaces the other strand of the duplex, resulting in the formation of a displacement loop, or D-loop. S. cerevisiae RAD52 mediates the exchange of RPA for RAD51, promoting RAD51 filament formation on RPA-coated ssDNA and stimulating RAD51 strand invasion. BRCA2 performs this mediator function in humans, leaving the role of human RAD52 unclear. Moreover, RAD52 mouse knockouts show little phenotype nor sensitivity to DSB-inducing agents, hRAD52 is inefficient at displacing RPA and stimulating strand exchange in vitro, and hRAD52 is not essential for RAD51 function or HR.

In summary, homologous recombination is important in the repair of DNA double-strand breaks, including those resulting from errors in DNA replication. How cells choose which pathway of DSB repair to use is under complex regulation. Mutations in proteins involved in homologous recombination lead to an increased risk in certain cancer types, highlighting the importance of this pathway. In the next section, we describe the RPA protein complex and its phosphorylation. This protein is involved in many DNA processes in cells, including DSB repair through HR.