Abstract
DNA damage provokes activation of cell cycle checkpoint response allowing time for DNA repair to maintain genomic integrity. During DNA damage response and repair, the chromatin is transiently disrupted to overcome the structural barrier and provide repair machinery access to damaged sites. Thus, restoration of functionally intact chromatin structure following DNA damage processing is crucial for retaining genetic as well as epigenetic information coded within modified histones. Our recent research has delved into how chromosomal incorporation of ubiquitinated histone H2A (uH2A) occurs following damage repair and the characteristic of this process in the context of nucleotide excision repair (NER) sub-pathways of global genomic repair (GGR) and transcription-coupled repair (TCR). We demonstrate that UV-induced uH2A foci formation occurs at cyclobutane pyrimidine dimer (CPD) sites in cells lacking XPC, DDB2, CSA or CSB, but not in cells lacking XPA, XPG or XPF. This repair-proficient and -deficient cell response indicated that uH2A incorporation strictly relies on successful damage repair occurring through either GGR or TCR sub-pathway. In contrast, XPA, XPG or XPF were not required for the formation of gH2AX foci in asynchronous cells, affirming this event as a pre-incision process. Notably, the H2A ubiquitin ligase Ring1B, a component of Polycomb repressor complex 1, did not localize at DNA damage sites. However, histone chaperone Caf-1 showed distinct localization to the damage sites. Knockdown of Caf-1 p60 abolished Caf-1 as well as uH2A foci formation. Caf-1 p150 was found to associate with NER factors TFIIH, RPA p70 and PCNA in chromatin. Overall data demonstrate that prompt Caf-1-mediated chromatin restoration following NER puts uH2A marks at the sites of repaired damage within chromatin. Our observations on CPD repair, and recent report on acetylated histone H3 driving the chromatin assembly after DSB repair, unfolds a novel phenomenon related to DNA repair-mediated alterations of epigenetic information within chromatin.