In order to survive, all organisms have evolved various mechanisms for repairing damaged DNA. In the case of double-stranded breaks (DSBs), homologous recombination (HR) is a critical repair mechanism that uses homologous DNA as a template for repair. Failures in mitotic recombination can lead to chromosomal rearrangements and cancer in humans, as seen in BRCA1 and BRCA2 mutations and failures in meiotic recombination can lead to infertility, miscarriage, and aneuploidy disorders, such as Downs Syndrome. Upon formation of a DSB, the damaged DNA is processed through a series of steps eventually leaving single-stranded DNA overhangs covered in a filament of proteins called recombinases. These recombinases then search the genome to find a homologous sequence, which can then be used as a template for repair of the damaged DNA. Eukaryotes have two recombinases, Rad51 and Dmc1. Rad51 is the only recombinase during mitotic HR, whereas, Dmc1 is expressed exclusively during meiosis. It is not known why most eukaryotes have two recombinases. However, a recent paper published in this lab demonstrated the first biochemical/biophysical difference between Rad51 and Dmc1. When presented with a mismatch during homologous DNA pairing, Dmc1 seems to stabilize the mismatch, while Rad51 appears to destabilize the mismatch. This differential response may reflect the unique biological roles of each recombinase: Rad51 is responsible for mitotic HR, and typically utilizes an identical sister chromatid as a template for repair; In contrast, Dmc1 must utilize homologs of different parental origins for meiotic HR. We propose that the ability of Dmc1 to stabilize mismatches reflects a requirement to promote recombination between template bearing single nucleotide polymorphisms during meiosis. In this proposal, we want to understand what are the structural elements that allow Rad51 and Dmc1 to behave differently to mismatches. We will identify DNA-binding regions of the two recombinases, determine amino acids that are uniquely conserved within each of the recombinases and swap these elements in order to make chimeric proteins. We will test whether these chimeric proteins produce the opposite response to mismatches as compared to their wild type forms. In the proposal, I have already demonstrated that I can create a Rad51 chimera that can stabilize mismatches and will attempt to create a Dmc1 chimera that can destabilize mismatches. I will then address the biological significance of mismatch (de)stabilization by incorporating my chimeras into yeast genomes and monitoring mitotic and meiotic HR in vivo. This proposal will attempt to provide further insight into and significance of the fidelity of eukaryotic recombination.
Homologous recombination (HR) is an essential pathway for organisms to accurately repair DNA damage. In humans, improper mitotic HR, such as in individuals with BRCA1 and BRCA2 mutations, can cause chromosomal rearrangements and cancer and improper meiotic HR can lead to infertility, miscarriage, and trisomy. This investigation will use biophysical, biochemical, and genetic approaches to understand the mechanistic basis for homologous recombination and will also seek to establish the key mechanistic difference between mitotic and meiotic recombination.
| Zhao, Weixing; Steinfeld, Justin B; Liang, Fengshan et al. (2017) BRCA1-BARD1 promotes RAD51-mediated homologous DNA pairing. Nature 550:360-365 |
| Lee, Ja Yil; Steinfeld, Justin B; Qi, Zhi et al. (2017) Sequence imperfections and base triplet recognition by the Rad51/RecA family of recombinases. J Biol Chem 292:11125-11135 |
| Ma, C J; Steinfeld, J B; Greene, E C (2017) Single-Stranded DNA Curtains for Studying Homologous Recombination. Methods Enzymol 582:193-219 |
