Lab Research
Protein Biochemistry
- Expression, purification, and characterization of DNA repair factors with emphasis on biochemical and biophysical analysis of protein-protein and protein-nucleic acid interactions
Double-Strand Break Repair
- Reconstituted biochemical systems to assess homologous recombination processes, including DNA end resection, synaptic complex formation, and RAD51-mediated strand invasion (D-loop)
Replication Fork Biology
- DNA fiber analysis, in situ protein interaction with nascent DNA replication forks (SIRF), fork regression and protection assays, break-induced replication
R-Loop Biology
- Cell-based detection of R-loops, in vitro resolution of R-loops
Cancer Biology
- Mutational analysis using patient-derived tumors to assess functional deficiency of recombinant proteins
Research
DNA becomes damaged upon exposure of cells to high-energy radiation and mutagenic chemicals prevalent in our environment. These agents, along with replicative stress and, endogenous oxygen radicals, and reactive aldehydes, pose a constant threat to the maintenance of a stable genome. Of the myriad DNA lesions, the DNA double-strand break (DSB) is among the most harmful because of its potential to cause deletions and other gross chromosome rearrangements. Accordingly, failure to properly resolve DSBs can lead to cellular transformation, cancer, and other pathologies, such as intellectual impairment and neurological disorders. Dysregulation of DSB repair pathways is a significant cause of innate and acquired resistance to cancer therapy as well.
The elucidation of DSB repair mechanisms will majorly impact health sciences. It will also provide insights to guide protection against DNA damage, explain drug resistance in cancer therapy, and identify new targets for developing novel therapeutics tailored to the DSB repair status in cancer patients.
Our laboratory investigates the mechanistic underpinnings of DSB repair mechanisms, particularly in the homology-directed DNA repair (HDR) pathway, which plays a critical role in the error-free elimination of DSBs and in replication fork repair (Figure below). Our research harnesses various biochemical, biophysical, and cell biological analytical tools to interrogate HDR factors at the molecular and cellular levels and draw a comprehensive picture of the HDR machinery. Currently, we are actively working on the following projects;
Regulation of DSB repair pathway choice:
Germline or acquired mutations in BRCA1 (Breast Cancer Gene 1) are clinically associated with the development and progression of breast and ovarian cancers. Cells deficient in BRCA1 or harboring mutant versions of the protein are defective in HR due to impaired DNA end resection and an inability to load the recombinase RAD51 onto ssDNA derived from resection. In contrast, loss of anti-resection factors, including 53BP1 and its interacting partner RIF1, leads to a partial restoration of DNA end resection and the ability to conduct HR in BRCA1-deficient cells. However, this apparently favorable outcome of HR restoration paradoxically renders cells resistant to PARP inhibitors, the newest class of anti-cancer drugs that are efficacious against BRCA- and other HR-deficient breast cancers. How exactly does 53BP1 or RIF1 loss in BRCA1-deficient cells promote resistance to PARP inhibitors remains undefined.
Recently, the multi-subunit Shieldin complex, comprising SHLD1, SHLD2, SHLD3, and REV7/MAD2L2, has been found to be recruited by 53BP1-RIF1 to DSBs, where it ′shields′ the DNA ends from unregulated resection. As such, Shieldin facilitates the engagement of NHEJ as the default repair pathway by preventing the channeling of DSBs into HR. Studies in human cells show that loss of Shieldin impairs NHEJ and leads to a hyper-resection phenotype, as indicated by an increase in levels of phosphorylated RPA as well as RPA nuclear foci, a reliable surrogate marker for DNA end resection, whereas its overexpression impairs HR. Moreover, mutations in genes encoding the Shieldin subunits promote resistance to PARP inhibitors in BRCA1-deficient cells, implicating its likely importance in the management of BRCA1-mutant tumors. Therefore, delineating the mechanism of action of Shieldin in DSB repair pathway choice will yield insights into how this pathway choice is regulated in humans and significantly impacts breast cancer biology and DNA repair.
Processing of RNA/DNA hybrids and R-Loop resolution by Senataxin-BRCA1 axis:
Senataxin (SETX), an RNA-DNA helicase, is mutated in a juvenile type of amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia 2 (AOA2). Such neuropathies are believed to stem from defects in RNA processing and transcription termination functions of SETX. Spontaneous DSBs generated during transcription significantly threaten genomic stability in post-mitotic neurons. This is evident by the association of multiple neuronal disorders with genetic defects in DNA repair genes such as NBS1 and ATM.
Recent reports of the accumulation of RNA at DSBs and an RNA-templated HR process in post-mitotic neurons warrant further investigation of the relevance of RNA in DSB repair. SETX accumulates at DSBs in a transcription-dependent manner and is associated with BRCA1 and other DDR factors. SETX depletion leads to an increase in 53BP1 foci and chromosomal translocations via NHEJ. We are currently invested in determining the association of SETX and other proteins involved in RNA metabolism with the HR repair pathway.
Regulation of DNA end resection
Mechanism of tumor suppressor proteins BRCA1, BRCA2, PALB2, RAD51, and paralogs, and their associated proteins in HDR and DNA replication fork protection:
Mechanism of Fanconi anemia proteins in HR
Recruitment
We are constantly seeking outstanding postdoctoral fellows to work on cutting-edge research in our lab. Please send your CV, a one-page cover letter, and a list of three references to Dr. Sung.
For additional information, please contact our lab manager, Stephen Holloway (holloways@uthscsa.edu).
Patrick Sung, DPhil