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.