Site-specific Acetylation of Histone H3 Decreases Polymerase β Activity on Nucleosome Core Particles in Vitro

Abstract

Histone posttranslational modifications have been associated with changes in chromatin structure necessary for transcription, replication, and DNA repair. Acetylation is one of the most studied and best characterized histone posttranslational modifications, but it is not known if histone acetylation modulates base excision repair of DNA lesions in chromatin. To address this question, we generated nucleosome core particles (NCPs) containingsite-specificallyacetylatedH3K14orH3K56andmea-sured repair of uracil and single-nucleotide gaps. We find that H3K56Ac and H3K14Ac do not significantly contribute to removal of uracils by uracil DNA glycosylase regardless of the translational or rotational position of the lesions within NCPs. In repair of single-nucleotide gaps, however, the presence of H3K56Ac or H3K14Ac in NCPs decreases the gap-filling activity of DNA polymerase ␤ near the dyad center, with H3K14Ac exhibiting stronger inhibition. To a lesser extent, H3K56Ac induces a similar effect near the DNA ends. Moreover, using restriction enzyme accessibility, we detect no changes in NCP structure or dynamics between H3K14Ac-NCPs and WT-NCPs containing single-nucleotide gaps. Thus, acetylation at H3K56 and H3K14 in nucleosomes may promote alternative gap-filling pathways by inhibiting DNA polymerase ␤ activity.Eukaryotic DNA is organized into arrays of nucleosomes, which constitute the primary level of chromatin compaction. Vital DNA-templated processes, including transcription and DNA repair, are dependent on protein-DNA interactions, which are restricted by the histone proteins in nucleosomes. The nucleosome core particle (NCP) 3 consists of 147 bp of DNA wrapped ϳ1.7 times around a histone octamer composed of a tetramer of histones H3 and H4 ((H3-H4) 2 ) flanked by two heterodimers of histones H2A and H2B (H2A-H2B) (1). Accessibility of nuclear factors to occluded DNA in this stable, yet dynamic protein-DNA complex is regulated by multiple mechanisms, including the activity of chromatin remodeling complexes (2, 3) and the intrinsic, partial unwrapping of DNA ends from the histone octamer (4, 5).The rate of DNA unwrapping, also referred to as DNA breathing, in nucleosomes is dependent on DNA sequence and the posttranslational modification (PTM) status of the histones (6 -8

Unencumbered Pol β lyase activity in nucleosome core particles

“…To accurately determine the effects of translational positioning on the Pol β dRP lyase reaction, we used the 147-bp 601 DNA strong positioning sequence and the recombinant histone octamer from X. laevis ( Supplementary Figure S1A&B ), both of which are known to generate stable well-positioned NCPs. As shown in Supplementary Figure S1C , all substrates were well positioned, independent of the location of the lesion in agreement with previous studies ( 28 , 42 ). Additionally, restriction enzyme accessibility assays of undamaged NCP resulted in the expected occlusion of the HhaI restriction site ( Supplementary Figure S1D ), as shown before ( 28 , 42 ), further confirming the integrity of these designed NCPs.…”

Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity

“…Histone acetylation is often associated with increased nucleosomal DNA unwrapping, particularly at distal translational positions (Neumann et al 2009), which could explain the faster repair of 7meG lesions at these positions in highly acetylated nucleosomes. While slower repair near the nucleosome dyad among highly acetylated nucleosomes seems counterintuitive, a recent report suggests that H3K14ac and other histone acetylations (i.e., H3K56ac) inhibit DNA polymerase β activity during repair of lesions located near the dyad of nucleosome substrates in vitro (Rodriguez et al 2016). The opposite trend was observed for histone PTMs associated with the 3 ′ coding regions of yeast genes (e.g., H3K36me3), presumably because these histone PTMs are associated with histone deacetylation (Lee and Shilatifard 2007;Weiner et al 2015).…”

“…However, it is not clear to what extent nucleosomes affect BER in vivo, since nucleosome positioning in eukaryotic genomes is generally weaker than the positioning sequences used for in vitro studies (Mao et al 2017) and the presence of cellular chromatin-remodeling enzymes can facilitate BER in nucleosomes (Hinz and Czaja 2015;Rodriguez et al 2015). Moreover, nucleosomes in vivo are marked by different histone post-translational modifications, which could potentially alter repair efficiency (Rodriguez et al 2016).…”

A cassette of basic amino acids in histone H2B regulates nucleosome dynamics and access to DNA damage

“…We recently showed that the effects of histone site-specific acetylation on DNA gap-filling activity by Pol ␤ are dependent on the structural location of the DNA gaps (45). In this study, we wished to determine whether the increased nucleosome dynamics near the DNA ends in ⌬HBR-NCPs was sufficient to allow accessibility to occluded DNA gaps located near the DNA ends (Ϫ49), "midway" (Ϫ21), or near the dyad center (ϩ4).…”