The role of CENP-A in the response to DNA double-strand breaks

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Exposure of cells to environmental agents, such as radiation, heavy metals, air pollutants and mutagenic chemicals, generates DNA double-strand breaks (DSBs) and other chromosomal lesions. Such environmentally induced chromosomal lesions are tolerated and ultimately eliminated via a complex, conserved mechanism termed the DNA damage response (DDR). Our long-term goal is to elucidate the molecular crosstalk between kinetochore-mediated mitotic regulation and the DDR. In particular, we strive to define the role of centromere protein A (CENP-A), a histone H3 variant, as a key mediator of 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. Importantly, we and others have made the surprising finding that CENP-A also localizes to DNA 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. We hypothesize 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 structure and function of the complex formed by CENP-A, BUB1, and other proteins at 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 as other centromere proteins (CENP-N, CENP-T, and CENP-U) and BUB1, a spindle checkpoint component, are recruited to DSBs. We will systematically examine whether known kinetochore proteins are localized at the DSB sites by immunofluorescence (IF) microscopic analysis. Aim 2: Assess the role of the spindle checkpoint in delaying cell cycle progression in DSB repair. We hypothesize that DSB-induced pseudo/kinetochores can activate the spindle checkpoint to cause a delay in mitosis, allowing DNA repair. We will first determine whether spindle checkpoints are localized at DBS sites by IF. We will determine whether the mitotic delay induced by DSBs is reliant on spindle checkpoint components when the DNA damage checkpoint activities are absent. Aim 3: Examine whether neocentromeres are formed upon failure of DNA repair. Occasionally, CENP-A-containing loci may become intact neocentromeres, which would rescue chromosome fragments without centromeres by generating new chromosomes with neocentromeres as a survival mechanism. We will screen for neocentromeres after DSB induction when DNA repair or the DNA damage checkpoint is compromised, and we will examine whether neocentromere formation increases under these conditions.