Mechanisms and Regulation of Resection in DNA Damage Response

Deoxyribonucleic acid (DNA) encodes genetic information essential for cell survival and function. However, it is constantly under assault from endogenous and exogenous damaging agents that not only threaten our own survival but also affect the faithful transmission of genetic information to our offspring. Double-strand breaks (DSBs) are one of the most hazardous forms of DNA damage, which if unrepaired or improperly repaired could lead to plethora of systemic human diseases including cancer. To deal with this problem, cells have evolved with a mechanism called DNA damage response (DDR) to detect, signal, and repair the breaks by inducing multiple cellular events. Resection is one of the key processes of cellular response to DSBs damage and is essential for genome maintenance, cell survival, and tumor suppression. Resection involves selective nucleolytic processing of the 5’ strand DNA at DSB ends to generate 3’ ssDNA overhangs, which in turn control both DNA repair and checkpoint response to the damage. Checkpoints coordinate the damage repair to other cellular processes including cell cycle regulation and gene expression. Despite its critical importance, the biochemical mechanisms and regulation of DSB resection is still not completely understood. Genetic studies in yeasts have suggested two steps mechanisms of resection: initiation by CtIP and MRN (Mre11-Rad50-NBS1) complex and extension by Dna2 and Exo1. We took a multipronged approach to study the resection process and have determined new mechanisms and regulation of both initiation and extension pathways. Here, we report a novel mechanism for the initiation of resection at clean DSBs mediated by Dna2 endonuclease activity. Our results strongly suggest that resection of blocked and free DSB ends is initiated via distinct mechanisms. In addition, we have demonstrated that the extension of resection by Exo1 is regulated both positively and negatively by Poly(ADP-ribosyl)ation, a prominent posttranslational modification at the sites of DNA damage. Our results suggest that Poly(ADP-ribose) not only promote initial damage recruitment of Exo1 but also prevent unscheduled and improper extension of resection. These two separate studies demonstrating new mechanisms for both initiation and extension steps of resection provide some critical new insights into the cellular response to DSBs damage

CHFR is important for the first wave of ubiquitination at DNA damage sites

Protein ubiquitination plays an important role in activating the DNA damage response and maintain-ing genomic stability. In response to DNA double-strand breaks (DSBs), a ubiquitination cascade occurs at DNA lesions. Here, we show that checkpoint with Forkhead-associated (FHA) and RING finger domain protein (CHFR), an E3 ubi-quitin ligase, is recruited to DSBs by poly(ADP-ribose) (PAR). At DSBs, CHFR regulates the first wave of protein ubiquitination. Moreover, CHFR ubiquitinates PAR polymerase 1 (PARP1) and regulates chromatin-associated PARP1 in vivo. Thus, these results demonstrate that CHFR is an important E3 ligase in the early stage of the DNA damage response, which mediates the crosstalk between ubiquitination and poly-ADP-ribosylation

PCNA promotes processive DNA end resection by

Exo1-mediated resection of DNA double-strand break ends generates 30 single-stranded DNA over-hangs required for homology-based DNA repair and activation of the ATR-dependent checkpoint. Despite its critical importance in inducing the overall DNA damage response, the mechanisms and regulation of the Exo1 resection pathway remain incompletely understood. Here, we identify the ring-shaped DNA clamp PCNA as a new factor in the Exo1 resection pathway. Using mammalian cells, Xenopus nuclear extracts and purified proteins, we show that after DNA damage, PCNA loads onto double-strand breaks and promotes Exo1 damage association through direct interaction with Exo1. By tethering Exo1 to the DNA substrate, PCNA confers processivity to Exo1 in resection. This role of PCNA in DNA resection is analogous to its function in DNA replication where PCNA serves as a processivity co-factor for DNA polymerases