Research
Ubiquitin signaling in the DNA damage response
In response to DNA damage from environmental or endogenous sources, cells evoke an elaborate signaling network known as the DNA damage response (DDR). Defects in this response can lead to hereditary cancer syndromes and can underlie the genomic instability which is a hallmark of sporadic cancers. The DDR promotes genomic integrity by targeting hundreds of factors in diverse pathways ranging from DNA replication and repair to cell-cycle arrest, senescence, metabolism, and immune regulation. Execution of the DDR relies upon a dynamic array of protein modifications, with ubiquitination playing a central role. Our lab elucidates biochemical and genetic relationships between DDR factors to understand how they are integrated and collectively regulated through ubiquitin signaling.
Replication-coupled repair
Replication fork collapse can induce chromosome instability and mutagenic events that cause cancer. Organisms have therefore evolved pathways to stabilize stalled replication forks and to repair collapsed forks through processes such as homologous recombination (HR). We have demonstrated that the ubiquitin ligase RFWD3, which is mutated in the genomic instability syndrome Fanconi anemia, ubiquitinates the single-stranded DNA binding factor RPA to promote homologous recombination at stalled replication forks and replication fork restart (Mol Cell 2015b). RPA ubiquitination occurs on numerous sites within multiple RPA subunits, reminiscent of protein group sumoylation that promotes the recruitment of repair factors in yeast HR.
Replication fork reversal is an important mechanism to protect the stability of stalled forks. While multiple enzymes have been identified that can remodel forks, their regulation remains poorly understood. We have discovered a new function for RFWD3 in the regulation of fork remodeling (J Cell Biol 2023). We find that RFWD3 promotes PCNA polyubiquitination to recruit the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. Through the analysis of replication intermediates by electron microscopy, we find that RFWD3 promotes replication fork reversal in a ZRANB3-epistatic manner. Consistent with a role for RFWD3 in fork reversal, inactivation of RFWD3 in BRCA2-deficient cells rescues fork degradation and collapse in these cells, analogous to ZRANB3 inactivation.
Quantitative proteomics
Numerous ubiquitin ligases have been implicated in the DNA damage response, yet finding their substrates by simple binding techniques can be difficult due to weak substrate interactions. To circumvent this problem, we have utilized a quantitative proteomic approach to globally profile ubiquitination. Initially, we used this approach to identify substrates of Cullin- RING ubiquitin ligases (Cell 2011), which are involved in numerous DNA repair processes. Subsequently, we used it to uncover novel ubiquitination events directly stimulated by DNA damage (Mol Cell 2015a), demonstrating the vast breadth of ubiquitin signaling in the DDR. We are continuing to use innovative proteomic approaches to characterize novel and poorly understood ubiquitin ligases in DNA damage signaling pathways.
Targeted cancer therapy
Defects in the DNA damage response can render tumors dependent upon specific DNA repair pathways for survival. We seek to exploit these vulnerabilities to develop therapies that target cancers with particular DNA repair defects, and we are employing methods to translate our work for this purpose.