Regulation of genome replication, recombination, and stress response

Maintaining genome stability requires the intricate coordination of DNA replication, DNA repair, and the DNA damage response. We have developed multi-disciplinary approaches to investigate some of the complex DNA transactions and signaling in these processes that are poorly understood. Our areas of inquiry include the regulation of the replisome and replication forks, the control of homologous recombination intermediates, and dampening of the DNA damage response. Our findings have led to novel hypotheses and testing them will deepen our understanding of critical genome regulation strategies. DNA replication must cope with many types of template barriers. The coping mechanisms entail close collaboration between the replisome and many regulators. One of our long-term goals is to elucidate how various regulators dynamically modify replisome functions. We will apply novel strategies to identify replisome changes and determine how the highly conserved multi-functional Smc5/6 complex promotes replisome function. Another goal of our studies is to determine the control of replication forks stalled at programmed barriers within the ribosomal DNA. These sites suffer topological stress that can drive fork instability. We will investigate how cells maintain the stalled replication forks in the face of this challenge to complete replication. When replication forks stalled by barriers fail to recover, collapsed forks and unreplicated DNA gaps can be repaired by homologous recombination, generating repair intermediates such as Holliday junctions. Resolving such joint DNA structures by specialized cleavage enzymes completes the repair process and prevents DNA entanglement. These enzymes collaborate with a range of regulators to engender efficient repair; however, the molecular roles of many regulators remain unclear. It is our goal to elucidate the mechanisms underlying the roles of these regulators, including the functionally coupled Smc5/6 and Esc2. In addition. we will study Smc5/6, which ties together DNA replication and recombinational control, in molecular detail. Genomic stress caused by DNA replication and repair failure activates the DNA damage checkpoint. While activating this checkpoint is beneficial, its persistence is detrimental to growth. Dampening the DNA damage checkpoint is thus essential to counter such harmful effects, but its mechanisms are understudied. One of our research goals is to identify checkpoint dampening pathways and their licensing mechanisms. This line of study will provide insights into the dynamic control of the DNA damage checkpoint. Outcomes of our proposed studies will expand our view of interconnected genome replication, repair, and stress response processes and inform studies of diseases that are linked to the malfunction of these pathways.