We use frog egg extracts to study the molecular mechanisms underlying DNA replication and repair, and how these processes go awry in human disease. We are working on the following areas:
1. DNA cross-link repair. Our cells are constantly exposed to reactive compounds (e.g. endogenous aldehydes) that covalently link the two strands of DNA to each other, thus generating highly toxic inter-strand crosslinks (ICLs). The same compounds also covalently cross-link proteins to DNA, generating DNA protein cross-links (DPCs). While defects in ICL repair cause bone marrow failure and cancer, defective DPC repair appears to cause premature aging and liver cancer. We have recapitulated ICL and DPC repair in frog egg extracts and find that in both cases, repair is triggered when a DNA replication fork collides with the damage (Raschle et al., Cell 2008; Duxin et al., Cell 2014; Semlow et al., Cell, 2016). We are now studying how ICLs and DPCs are repaired in the context of replication and how defects in these repair reactions cause disease.
2. Single molecule studies of double-strand break repair. We have shown that DNA replication can be studied at the single molecule level in frog egg extracts (Yardimci et al., Mol. Cell, 2010; Fu et al., Cell 2011; Kochaniak et al., Nature Methods, 2012). In collaboration with Joe Loparo's lab, we are now using this approach to examine how DNA double strands breaks are repaired via homologous recombination and non-homologous end joining (Graham et al., Mol. Cell, 2014).
3. Replication termination. When two DNA replication forks converge at the end of replication, how is DNA synthesis completed and how is the replication machinery disassembled? We recently recapitulated site-specific replication termination in egg extracts, leading to a new and unexpected model of this process (Dewar et al., Nature, 2015). We are now using this approach to elucidate how removal of the replication machinery is regulated by the ubiquitin system (Dewar et al., G&D, 2017).
Seeley G Mudd Building, Room 204A
240 Longwood Ave.
Boston, MA 02115