Abstract
Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2–12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in ‘designed’ nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER.
Highlights
Base excision repair (BER) processes non-helix distorting lesions and is composed of two subpathways that differ in the number of nucleotides incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2–12 nts
In agreement with previous studies[14,15,16], we find that when the lesion is in the nucleosome core particle (NCP), DNA cleavage is significantly inhibited in (a) nucleosome substrates compared to naked DNA, (b) nucleosome substrates where the translational position of the uracil is closer to the DNA dyad, and (c) nucleosome substrates where the rotational position of the uracil is toward the histone octamer (Fig. 2b–e)
We identify a novel nucleosome-dependent bias for SP base excision repair (BER) when using bovine testis nuclear extracts (BTNEs) (Fig. 3b–f) or purified enzymes (Fig. 3g)
Summary
We identify a novel nucleosome-dependent bias for SP BER when using BTNE (Fig. 3b–f) or purified enzymes (Fig. 3g). When using a gap substrate, we determined that this 1-nt limit is imposed by the NCP in the absence of DNA ligase activity (Supplementary Fig. S3a–d), indicating DNA ligase activity is not the limiting factor in nucleotide extension in nucleosomes. Our Pol β-neutralization results indicate that Pol βis responsible for the majority of BER synthesis at gapped substrates in NCP DNA (Fig. 4), while substantial DNA polymerase activity is maintained in naked DNA when Pol βis neutralized. This provides further evidence that the extracts contain other polymerases to process THF nicks (Supplementary Fig. S5B; compare naked to NCP) These results suggest there is a selection for the DNA polymerase that carries out BER of gapped substrates at or near the NCP-linker junction (Fig. 5). As Pol βhas been shown to be evolutionarily linked to the development of metazoans[40], it is possible that this DNA polymerase evolved to repair compact DNA because it is less restricted in a compact chromatin environment than other DNA polymerases
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