The histone H3 variant, centromere protein A (CENP-A), also localizes to DNA at double-strand breaks (DSBs), implying involvement in responses to DNA damage. CENP-A is normally a constituent of centromere specific chromatin, which forms the foundation for assembly of the kinetochore, a proteinaceous structure comprising a number of conserved protein complexes, that serve as the interface between chromosomes and spindle microtubules. CENP-A plays a crucial role in centromere identity and kinetochore assembly, and is essential to centromere and kinetochore functions, but why it should be recruited to DSB foci is not known. Our working hypothesis is that CENP-A is recruited to DSBs to create a DNA damage-specific form of chromatin to which DNA-damage checkpoint/repair proteins are recruited. Based on this idea, we propose a new model to describe an important mechanism of survival of unrepaired DSBs. In this model, unrepaired DNA damage produces acentric chromosome fragments that may harbor essential genetic information. To stably inherit these acentric fragments, a neocentromere must be formed at the DSB site marked by CENP-A protein on the broken chromosome fragment. The neocentromere facilitates microtubule attachment and proper segregation during mitosis. We hypothesize that incorporation of CENP-A at a DSB site can result in the formation of a neocentromere, and survival of cells following the failure to repair DSBs induced by chemotherapeutic agents or radiation therapy. The specific objective of this proposal is to determine the role of CENP-A in the DDR.
Aim 1. Determine significance of CENP-A localization at DSB sites. We will introduce DSBs into chromosomes in cells depleted of CENP-A, then analyze DNA damage checkpoint activity and DNA repair kinetics at the DSB site, and determine if recruitment of known DNA damage checkpoint/repair proteins to DSB sites is abrogated. Furthermore, we will measure DNA repair activity, and survival in cells depleted of CENP-A.
Aim 2. Determine if neocentromere formation occurs at the site of DSBs. We hypothesize that a neocentromere or a pseudo-neocentromere assembles at the DSB site in a CENP-A-dependent manner. Some spindle assembly checkpoint components have been identified at DSBs, but it is unknown whether a functional kinetochore, which can capture microtubules, is assembled. To test this hypothesis, we will perform immunofluorescence microscopy to detect all the kinetochore components. We will also examine whether neocentromere formation is increased when DDR pathways fail. These studies will provide novel insight into the role of CENP-A in cell survival. The research project will establish a novel role of CENP-A in the DDR, and reshape the map of the DNA-damage signaling pathway. If neocentromere formation occurs at the DSB site, it will represent a previously unknown mechanism for protecting cells from genome instability in response to DNA damage.
Activities of DNA damage response pathways are crucial determinants of radiosensitivity or chemosensitivity of tumor cells. Therefore developing inhibitors targeting these pathways that can be used in combination with DNA-damaging agents will prove efficacious therapeutic strategies with fewer side effects. We will determine the role of centromeric chromatin component, CENP-A in DNA damage response. Our studies will identify previously undescribed crosstalk between regulation of chromosome segregation and DNA damage response, thereby contributing to identification of new molecular targets for anticancer therapies.
|Niikura, Yohei; Kitagawa, Risa; Kitagawa, Katsumi (2016) CENP-A Ubiquitylation Is Inherited through Dimerization between Cell Divisions. Cell Rep 15:61-76|