The DNA replication-repair interface: mechanisms and regulation

PROJECT SUMMARY DNA replication in eukaryotes is carried out multiprotein complexes that carry out a variety of enzymatic activities required for faithful genome duplication. Regulation and coordination of these activities at replication forks are essential for genomic stability. The replisome regularly encounters impediments that stall the replication fork, including DNA damage, aberrant DNA structures, and transcription conflicts. Stalled forks are a major source of genomic instability and underlie diseases including cancer. Replication-repair pathways serve to stabilize, repair, and restart stalled forks. However, the molecular mechanisms of these pathways are poorly understood. We are addressing this gap in knowledge by determining structures and mapping interactions within and among multiprotein-DNA complexes and combining this information with their biochemical and cellular functions. Our long-term goal is to decipher the molecular mechanisms and regulation of replication-repair pathways to understand how errors in these processes lead to disease. We are currently focused on three enzymatic activities essential for faithful genome duplication—(1) initiation of DNA synthesis by DNA polymerase (pol) α–primase, (2) fork reversal and template switching by ATP-dependent DNA translocases, and (3) DNA glycosylase mediated repair of interstrand DNA crosslinks (ICLs). (1) Polα–primase is a core component of the eukaryotic replisome that initiates DNA synthesis by generating chimeric RNA-DNA primers necessary for replicative synthesis by polδ and polε. Despite the importance of this critical activity, how polα–primase primes synthesis in the context of the replisome and how the primer is transferred to polδ for Okazaki fragment synthesis are unknown. We are determining structures of polα–primase with nucleic acid substrates and protein partners at various stages of its catalytic cycle and visualizing conformational states by electron microscopy and biophysical approaches. (2) Fork reversal involves remodeling a stalled fork into four-way junctions to prevent fork collapse and facilitate replication restart. Our work will address how HLTF, SMARCAL1, and FBH1 provide unique repair activities at damaged forks, their mechanisms of fork reversal, and how the ubiquitin ligase activities of HLTF and FBH1 regulate fork reversal. (3) Abasic (AP) sites are one of the most abundant forms of DNA damage and react with the opposite strand to form an AP-ICL. The NEIL3 glycosylase unhooks AP-ICLs at convergent replication forks by an unknown mechanism. Our previous work defined NEIL3’s specificity for AP-ICLs in a particular arrangement and spacing from a fork junction, as well as the structure and DNA binding of the C- terminal GRF zinc finger domain. We are working to understand how the unique architecture of NEIL3 enables its recruitment and positioning at convergent forks, and how the catalytic domain is capable of accessing a conformationally constrained crosslinked nucleotide. Our work addresses critical gaps in knowledge related to how the intrinsic activities of these enzymes are regulated by interactions within the replisome.