Beyond DNA end resection: Role of homologous recombination in the maintenance of genome stability
DNA of all living organisms to prone to damage. DNA breaks can occur either directly or indirectly upon exposure to intermediates of cellular metabolism, drugs or radiation. To restore DNA integrity, cells possess non-homologous end joining (NHEJ) and homologous recombination (HR) pathways. Whereas NHEJ is template independent and therefore often mutagenic, HR is template-directed and thus largely accurate. My laboratory is specifically interested in the mechanisms of homologous recombination. We primarily use a biochemical approach, where we express and purify recombinant proteins that catalyze elements of the recombination pathway, and reconstitute the reactions with synthetic DNA substrates in vitro. Studying the enzymatic reactions in a controlled system allows us to unambiguously determine underlying molecular mechanisms. My laboratory has been very interested in the first steps of the recombination pathway, which involves nucleolytic processing (resection) of the 5'-terminated DNA strand at DNA break sites, leading to 3' overhangs. This process commits the DNA double-strand break (DSB) repair to the HR pathway and prevents NHEJ. DNA end resection is initiated by the MRX-Sae2 complex in yeast and by MRN-CtIP in humans; the biochemical characterization of these factors is a subject of my ongoing ERC grant. Here, I propose to determine mechanisms of processes occurring downstream of the initial DNA end resection by the MRX/N complex, which includes the following three aims (see Fig. 4 for graphical overview):Aim I. DNA end resection by the Dna2 nuclease: functional interaction with replication protein A (RPA). MRN/X nuclease resects DNA only in the immediate vicinity of the DNA break. Longer lengths of DNA may be subsequently resected by the Dna2 helicase-nuclease, which is regulated by the single-strand DNA binding protein RPA. We aim to determine the molecular mechanism underlying the RPA stimulatory effect on Dna2.Aim II: Activation of DNA damage checkpoint by DNA end resection machinery. The presence of DNA ends as well as ssDNA resulting from DNA end resection recruits proteins that activate DNA damage checkpoint signaling kinases ATM/Tel1 and ATR/Mec1. This includes DNA end resection nucleases, including the MRX/N protein complex, but also Dna2 in yeast. We aim to determine how DNA end resection regulates DNA damage signaling. Aim III: Recombination in DNA replication: Regulating the RAD51 recombinase. Downstream of DNA end resection and initial checkpoint signaling, the RPA protein coating ssDNA is replaced by the strand exchange protein RAD51, which catalyzes homology search for template DNA. This process has been largely defined in DSB repair. However, the primary role of HR appears to be to repair DNA damage arising during DNA replication. One of the functions of RAD51 may be to help catalyze the formation of reversed replication forks, and to protect DNA at stalled forks from degradation. This however remains mechanistically undefined. Here we will determine how is the RAD51 function regulated upon replication fork stalling. This includes investigation of the MMS22L-TONSL complex and other factors, which promote DNA stability during replication. These experiments will better define how homologous recombination repairs broken DNA, as well as how it assists in replication of damaged DNA. We will also determine how recombination proteins signal the presence of DNA damage to the checkpoint signaling cascade. These functions are essential to protect us from genome instability and tumorigenesis.