Histone H4 LRS mutations can attenuate UV mutagenesis without affecting PCNA ubiquitination or sumoylation.

UV is a significant environmental agent that damages DNA. Translesion synthesis (TLS) is a DNA damage tolerance pathway that utilizes specialized DNA polymerases to replicate through the damaged DNA, often leading to mutagenesis. In eukaryotic cells, genomic DNA is organized into chromatin that is composed of nucleosomes. To date, if and/or how TLS is regulated by a specific nucleosome feature has been undocumented. We found that mutations of multiple histone H4 residues mostly or entirely embedded in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain attenuate UV mutagenesis in Saccharomyces cerevisiae. The attenuation is not caused by an alteration of ubiquitination or sumoylation of PCNA (proliferating cell nuclear antigen), the modifications well-known to regulate TLS. Also, the attenuation is not caused by decreased chromatin accessibility, or by alterations of methylation of histone H3 K79, which is at the center of the LRS surface. The attenuation may result from compromised TLS by both DNA polymerases ζ and η, in which Rad6 and Rad5 are but Rad18 is not implicated. We propose that a feature of the LRS is recognized or accessed by the TLS machineries either during/after a nucleosome is disassembled in front of a lesion-stalled replication fork, or during/before a nucleosome is reassembled behind a lesion-stalled replication fork.

Histone H4 H75E mutation attenuates global genomic and Rad26-independent transcription-coupled nucleotide excision repair.

Nucleotide excision repair (NER) consists of global genomic NER (GG-NER) and transcription coupled NER (TC-NER) subpathways. In eukaryotic cells, genomic DNA is wrapped around histone octamers (an H3-H4 tetramer and two H2A-H2B dimers) to form nucleosomes, which are well known to profoundly inhibit the access of NER proteins. Through unbiased screening of histone H4 residues in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain, we identified 24 mutations that enhance or decrease UV sensitivity of Saccharomyces cerevisiae cells. The histone H4 H75E mutation, which is largely embedded in the nucleosome and interacts with histone H2B, significantly attenuates GG-NER and Rad26-independent TC-NER but does not affect TC-NER in the presence of Rad26. All the other histone H4 mutations, except for T73F and T73Y that mildly attenuate GG-NER, do not substantially affect GG-NER or TC-NER. The attenuation of GG-NER and Rad26-independent TC-NER by the H4H75E mutation is not due to decreased chromatin accessibility, impaired methylation of histone H3 K79 that is at the center of the LRS domain, or lowered expression of NER proteins. Instead, the attenuation is at least in part due to impaired recruitment of Rad4, the key lesion recognition and verification protein, to chromatin following induction of DNA lesions.