Our long-term goal is to elucidate the molecular crosstalk between kinetochore-mediated mitotic regulation and the DNA damage response (DDR). In particular, we strive to define the role of centromere protein A (CENP-A), a histone H3 variant, in this crosstalk. CENP-A is a constituent of the centromere-specific chromatin essential for the assembly of the kinetochore, a proteinaceous structure that provides the connection between chromosomes and spindle microtubules. CENP-A plays a crucial role in centromere identity and kinetochore assembly. Overexpression of CENP-A causes aneuploidy, and CENP-A is highly expressed in several cancers. Importantly, we and others have made the surprising finding that CENP-A also localizes to DNA double-strand breaks (DSBs) in normal and immortalized human and mouse cells. The available evidence suggests that CENP-A functions in DSB repair, but the mechanism by which it accomplishes this feat remains to be determined. Our objective here is to determine the function of CENP-A in the cellular response to DSBs. We hypothesize (i) that CENP-A mediates key functions at DSBs for proper DDR, and (ii) that CENP-A nucleates the formation of a pseudo/kinetochore at DSB sites to activate the spindle checkpoint and delay cell cycle progression when DNA damage repair fails.
We propose the following Specific Aims to test our hypothesis:
- Aim 1. Determine the functional significance of CENP-A localization at DSB sites during DDR. Our working hypothesis is that CENP-A at DSB sites is required for proper DDR. We will identify CENP-A-associated proteins that interact with CENP-A in response to DSB. Based on the results, we will determine the role of CENP-A in DDR. 1) Identify protein partners of CENP-A in response to DSB induction. We have identified 10 such DSB-induced interactors of CENP-A. We will determine the DDR role of each protein together with CENP-A. 2) Examine whether CENP-A is involved in the non-homologous end-joining (NHEJ) pathway. 3) Determine whether CENP-A is involved in DNA damage checkpoint activity induced by DSBs. 4) Investigate the role of R-Loops in CENP-A deposition at DSB sites. 5) Examine CENP-A and HJURP for R-loop binding.
- Aim 2. Determine the role of CENP-A in the activation of the spindle checkpoint in response to failed DNA damage repair. How can the spindle checkpoint be activated by DSBs? Our working hypothesis is that a CENP-A-containing complex forms a “pseudo kinetochore” that assembles at DSBs whereupon it activates the spindle checkpoint (which monitors kinetochore-microtubule attachment) when DDR fails to eliminate the DNA lesions in a timely fashion. This hypothesis is based on evidence that other centromere proteins (CENP-N, CENP-T, and CENP-U) and BUB1, a spindle checkpoint component, are present at DSBs. 1) Determine the structure and function of the complex formed by CENP-A, BUB1, and other proteins at DSBs. 2) Assess the role of the spindle checkpoint in delaying cell cycle progression in DSB repair. 3) Examine whether neocentromeres are formed upon failure of DNA repair.
Our research is focused on understanding the role of this core centromere-specific chromatin component in the cellular response to DNA damage. We will determine why cells divert this centromeric histone to the DDR, which represents a major unanswered question in the DDR field. Specifically, the proposed studies will determine whether the deposition of CENP-A at DSB sites is required for spindle checkpoint activation when break repair is slowed or fails and whether this is accomplished via the assembly of a neo-kinetochore at DSB sites. The completion of our project will define a novel role for CENP-A in DDR and reshape our understanding of DNA-damage signaling that helps compensate for suboptimal DNA repair. If pseudo/kinetochore formation occurs at DSB sites, it would suggest a novel mechanism for protecting cells from genomic instability in response to DNA damage.
The PI is a graduate faculty member in the Integrated Biomedical Science Graduate Program at The University of Texas Health San Antonio (UTHSA). The proposed projects will provide educational and research experience for postdoctoral, graduate, undergraduate and high school students in the San Antonio area. Most of these students are Hispanic and many are underprivileged. The PI will integrate interested students into his mentoring research team to conduct their research in his laboratory. In addition, UTHSA offers various professional development workshops/sessions on campus to encourage postdoctoral fellows in establishing their own careers in scientific research. In addition, students from underrepresented minorities in science, technology, engineering, and mathematics (STEM) will participate in the proposed research and educational activities. UTHSA is a Hispanic-serving institution and works closely with the University of Texas-San Antonio undergraduate campus to foster student research opportunities. The 12-week CURE program brings local minority undergraduates to the UTSA labs each summer for hands-on experiences. Also, the BEAT Academy program is a 1-week outreach program for local high school students to explore careers in medical research. Via these established programs, the PI can recruit 4-6 students each year into his lab with stipends provided by the programs. Research findings will be regularly disseminated at relevant meetings and published in peer-reviewed journals.