Simon Powell: BRCA1 Superfamily and Their Interactions

The BRCA1 superfamily are Rad9-like proteins containing C-terminal BRCT repeats that assist in the binding of phosphorylated proteins involved in the DNA damage response and DNA repair. These proteins, BRCA1, MDC1, and 53BP1, have different functions ascribed to their core and N-terminal domains, which contribute to the specific function of each protein. Key post-translational modifications are controlled by these proteins.

Post-translational Modifications That Regulate Homologous Recombination

Different stages in the engagement of homologous recombination can be identified. The earliest steps in the DNA damage response are responsible for recruiting MDC1, 53BP1, and BRCA1 to sites of DNA damage and replication-associated lesions. Many of the post-translational modifications have been described in the extensive literature in this field over the past five years. The ATM- and Chk2-dependent modifications of BRCA1 have been characterized to determine cell-cycle-checkpoint and recombinational repair functions of the protein, respectively. 53BP1 appears to have negative regulation of BRCA1-dependent functions in DNA repair, and we are investigating whether this functions by regulating Chk2 activity at DNA damage sites. Chk2 phosphorylates BRCA1 at serine-988, which appears to be a critical step for activating homologous recombination. The molecular mechanisms involved in this control step are under investigation.

Does Chk2 Phosphorylation of BRCA1 Regulate CtIP Activity?

CtIP, the CtBP (carboxy-terminal binding protein) interacting protein is also a binding partner of BRCA1 and is involved in the processing of DNA damage for repair by homologous recombination (HR). In response to DNA damage, BRCA1 phosphorylation on S988 by Chk2 is important for HR. The BRCA1-S988A mutant is capable of being recruited to DNA damage and signaling the cell-cycle checkpoints in S and G2, but its function in HR is markedly impaired. Elevated levels of MRE11 foci were observed in the BRCA1-S988A mutant cells in comparison to wild type. CtIP and MRE11 form a complex at DSBs, which is required for effective DNA end resection during HR repair. Therefore, we are asking whether BRCA1-S988 phosphorylation is required for the association of CtIP, DNA end resection prior to HR, and turnover of CtIP once resection is complete.

Requirements for Cleavage of Blocked Replication Forks

Using plasmids containing Ter sequences, we can observe Tus-protein-induced replication block in human cells. We are investigating cleavage of the blocked replication fork in rescued plasmids. The approach we have taken to identify cleavage is to use ligation-mediated PCR, which has been used to monitor breakage in VDJ recombination as well as other applications. A double-stranded oligonucleotide, designed to ligate to any cleaved duplex, is ligated by end filling (using Klenow fragment polymerase) and blunt-end ligation. The reaction is designed to function using the rescued plasmids following 24 to 48 hours of processing in human cells. For the PCR component of the reaction, either strand of the oligonucleotide could serve as 5’-3’ primer of one component of the PCR reaction, depending on the orientation of the ligation. Away from the presumed site of breakage, second primer sequences oriented toward the break are used to observe evidence of cleavage of the plasmid template. Genetic determinants of replication fork cleavage are under investigation.