Xenopus CENP-A assembly into chromatin requires base excision repair proteins

Abstract

CENP-A is an essential histone H3 variant found in all eukaryotes examined to date. To begin to determine how CENP-A is assembled into chromatin, we developed a binding assay using sperm chromatin in cell-free extract derived from Xenopus eggs. Our data suggest that the catalytic activities of an unidentified deoxycytidine deaminase and UNG2, a uracil DNA glycosylase, are involved in CENP-A assembly. In support of this model, inhibiting deoxycytidine deaminase with zebularine, or uracil DNA glycosylase with Ugi, uracil or UTP results in a lack of detectable CENP-A on sperm DNA.Conversely, inducing DNA damage increases the level of CENP-A detected on sperm chromatin. Our data suggest that base excision repair may be involved in assembly of this histone H3 variant.3

Centromeres: The wild west of the post-genomic age

“…Proteolytic "cleanup" could explain why the number of CENP-A foci on Xenopus sperm, initially higher than expected, decreases down to the expected number of 1 per chromosome. 46 This would also be consistent with protein degradation by ubiquitin-dependent mechanisms after DNA repair. [103][104][105] CENP-A is abruptly lost about an hour after accumulating at the site of DNA damage in human cells.…”

“…[88][89][90] An additional dimension is added by evidence that cytidine deamination and uracil removal are required for CENP-A assembly. 46 This model is analogous to cytidine deamination and uracil removal in B cells. 87 In this model, cytidine deamination in repetitive class-switch regions produces uracil, which is removed by the highly conserved nuclear uracil DNA N-glycosylase UNG2, leading to DNA breaks (Fig 1).…”

“…However, other evidence demonstrates that CENP-A also binds to damaged DNA outside of centromeres. 46,47 CENP-A can be recruited throughout interphase by double-strand breaks. 47 This finding could reconcile prior disputes about timing.…”

“…Using quantitative image analysis, it was demonstrated that inhibiting DNA replication of Xenopus sperm DNA in egg extracts caused both the size and incidence of CENP-A foci to increase. 46 This unexpected finding raised the possibility that CENP-A could be recruited by DNA damage at stalled replication forks. This result implied that, contrary to the prevailing epigenetic model, CENP-A assembly does not require a preexisting template.…”

“…46,47 several DNA repair proteins have been found at endogenous centromeres, and/or associated with CENP-A by either genetic or biochemical means ( Table 1). Rather than being impervious to DNA damage, the constant presence of proteins involved in DNA repair should instead make centromeres highly proficient at DNA repair.…”