The laboratory of Dr. Stark investigates the regulation and fidelity of chromosomal break repair pathways. The long-term goal of his laboratory is to understand the factors and conditions that affect the regulation and fidelity of chromosomal break repair in mammalian cells. This goal is important to understand how chromosomal rearrangements can arise from such repair, which influences both to the etiology of cancer, as well as cancer cell response to clastogenic therapeutics. In one of their central projects, using reporter systems for the end-joining (EJ) repair pathway, they seek to define the role of several DNA repair factors on controlling the fidelity of EJ, which is critical to limit genome rearrangements, and is likely important for resistance to radiotherapy. Related projects are focused on examining the homologous recombination (HR) repair pathway. For one, Stark’s group is seeking to define genes that affect the requirement for the breast and ovarian cancer tumor suppressor gene BRCA1 during HR, with a particular focus on the RNF168/53BP1 pathway. In addition, they seek to develop functional biomarkers for loss of the HR pathway using ovarian epithelial carcinoma cultures, with a goal of improving our understanding of how such loss of this pathway affects therapeutic response to clastogens in ovarian cancer.
The Stark lab aims to include scientific trainees at several stages, including post-baccalaureate research associates (RA1/2), PhD candidates in the Irell and Manella Graduate School of the City of Hope, and postdoctoral fellows.
The Regulation and Fidelity of Chromosomal Break Repair Pathways
While high-fidelity repair of DNA damage is essential for genome maintenance, individual DNA repair pathways can be prone to cause genetic loss. The long-term objective of our lab’s research is to understand the factors and pathways that influence the extent of genetic loss during repair of chromosomal breaks in mammalian cells. This objective is important for an understanding of the process of mutagenesis during cancer development, as well as for a mechanistic characterization of potential targets of drugs that could increase the efficacy of cancer treatments that utilize DNA damaging agents.
As one approach to this objective, we are studying the mechanism of homologous repair of a break generated by the rare cutting endonuclease, I-SceI. Specifically, we generate a mammalian cell line that contains an I-SceI site integrated into the chromosome in the context of a reporter gene, express I-SceI in the cells, and allow the cells to repair the break. We then monitor the different products of such repair, which are associated with varying degrees of genetic loss. Furthermore, we are testing how disruption of individual genetic factors influences the efficiency of individual repair products.
For example, we found that disruption of RAD51 function by expression of a dominant-negative peptide, RAD51-K133R, resulted in a decrease in the efficiency of a precise form of repair, gene conversion, and an increase in two mutagenic forms of repair, single-strand annealing and extensive gene conversion. In addition, we found that the breast cancer susceptibility factors, BRCA1 and BRCA2, differentially affected these pathways. Disruption of BRCA2 caused a similar result as disruption of RAD51, in that the efficiency of gene conversion was decreased and single-strand annealing was increased. In contrast, disruption of BRCA1 caused a decrease in the efficiency of both gene conversion and single-strand annealing.
Recently, we have been analyzing a pathway of repair that results in deletion mutations, often with evidence of short homologous sequences at the repair junctions (alt-NHEJ). Such repair events mimic a variety of oncogenic genome rearrangements. We have begun to study the genetic factors that affect these events, and have found that KU/CtIP appear to affect the initial stages of repair of both alt-NHEJ and recombination. However, these pathways appear to diverge at later steps, as relates to the role of the repair factors RAD52, ERCC1, and RAD51. We are working to further characterize the mechanisms of these factors and pathways of chromosome break repair, in order to improve our understanding of the process of genetic loss during cancer development and treatment.
PhD candidate Meilen Munoz, M.S. Cal Poly Pomona
PhD candidate Sean Howard, B.S. University of Iowa
Postdoctoral fellow Dr. David Onyango, PhD Indiana University School of Medicine.
Post-baccalaureate RA1 Ms. Diana Yanez, B.S. UCLA
Ms. Anita Cheng
Mr. Nick Huang
Dr. Nicole Bennardo (graduated 2011)
Dr. Amanda Gunn (graduated 2011)
Dr. Corentin Laulier