ABSTRACT
The highly charged histone N-terminal domains are engaged in inter- and intra-nucleosomal interactions, and contain a host of sites used for posttranslational modification. We have studied the effect of deleting residues 30-37 from the N-terminal domain of histone H2B in yeast cells, on nucleotide excision repair (NER) following UV irradiation, as these cells are quite sensitive to UV. We find that H2B Delta30-37 cells exhibit reduced NER efficiency at three specific chromatin loci: the transcriptionally active, RPB2 locus; the transcriptionally silenced, nucleosome-loaded HML locus; and the transcriptionally repressed, non-silenced, GAL10 locus. Nuclease digestion studies indicate that H2B Delta30-37 chromatin has increased nucleosome accessibility and/or nucleosome mobility. In addition, H2B Delta30-37 mutants acquire more DNA damage, compared to wt cells, following the same dose of UV radiation. Reducing the level of damage in H2B Delta30-37 cells to match that of wt cells restores the NER rate to wt levels in the RPB2 and GAL10 loci, but NER efficiency remains low in the silenced HML locus. Interestingly, recruitment of Snf5 to the HML locus is reduced in H2B Delta30-37 cells and more transient following UV irradiation. This may reflect a lower binding affinity of the SWI/SNF complex to H2B Delta30-37 nucleosomes.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Histones/chemistry , Transcription Factors/metabolism , Amino Acids/chemistry , Chromatin/chemistry , DNA Damage , Gene Silencing , Histones/genetics , Micrococcal Nuclease , Mutation , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence Deletion , Ultraviolet Rays , Yeasts/genetics , Yeasts/metabolism , Yeasts/radiation effectsABSTRACT
DNA acts as a 'workbench' for various nuclear processes that occur inside living cells. In eukaryotic cells, DNA is highly compacted in a structural hierarchy with histones and other proteins into chromatin. This compaction affects DNA structure and coordinates the accessibility to site-specific nuclear factors during DNA processing events. DNA repair is no exception to this general rule and several reviews have appeared recently that discuss this topic in detail [1-3]. Here, we focus on recent findings correlating changes in DNA repair with subtle variations in the chromatin landscape.
Subject(s)
Chromatin Assembly and Disassembly , DNA Repair , Adenosine Triphosphate/metabolism , Chromatin/metabolism , DNA Damage , Histones/metabolism , Humans , Models, Genetic , Models, Molecular , MutationABSTRACT
Single amino acid changes at specific DNA contacts of histones H3 and H4 generate SWI/SNF-independent (Sin) mutants in yeast. We have analyzed the effect of the Sin mutation at R45 of histone H4 on cell survival following UV irradiation, nucleotide excision repair (NER) and chromatin structure. We find that this mutation renders yeast cells more resistant to UV damage and enhances NER at specific chromatin loci. In the transcriptionally silent HML, repressed GAL10 and the constitutively active RPB2 loci, H4 R45 mutants exhibit enhanced repair of UV-induced cyclobutane pyrimidine dimers (CPDs) compared to wild-type (wt). However, the H4 R45 mutation does not increase the transcription of NER genes, disrupt transcriptional silencing of the HML locus or alter repression in the GAL10 locus. We have further shown that the H4 R45C mutation increases the accessibility of nucleosome DNA in chromatin to exogenous nucleases and may expedite nucleosome rearrangements during NER. Taken together, our results indicate that the increased repair observed in Sin mutants is a direct effect of the altered chromatin landscape caused by the mutation, suggesting that such subtle changes in the conserved histone residues can influence the accessibility of DNA repair factors in chromatin.
Subject(s)
DNA Repair , Histones/genetics , Saccharomyces cerevisiae/genetics , Ultraviolet Rays , Adenosine Triphosphatases/genetics , Amino Acid Substitution , Cell Survival , Chromatin/metabolism , DNA Damage , Deoxyribonucleases/metabolism , Gene Deletion , Gene Expression , Gene Silencing , Histones/metabolism , Kinetics , RNA Polymerase II/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/radiation effects , Saccharomyces cerevisiae Proteins/genetics , Transcription, GeneticABSTRACT
The ABA responsive ABI3 and the auxin responsive ARF family of transcription factors bind the CATGCATG (Sph) and TGTCTC core motifs in ABA and auxin response elements (ABRE and AuxRE), respectively. Several evidences indicate ABI3s to act downstream to auxin too. Because DNA binding domain of ABI3s shows significant overlap with ARFs we enquired whether auxin responsiveness through ABI3s could be mediated by their binding to canonical AuxREs. Investigations were undertaken through in vitro gel mobility shift assays (GMSA) using the DNA binding domain B3 of PvAlf (Phaseolus vulgaris ABI3 like factor) and upstream regions of auxin responsive gene GH3 (-267 to -141) and ABA responsive gene Em (-316 to -146) harboring AuxRE and ABRE, respectively. We demonstrate that B3 domain of PvAlf could bind AuxRE only when B3 was associated with its flanking domain B2 (B2B3). Such strict requirement of B2 domain was not observed with ABRE, where B3 could bind with or without being associated with B2. This dual specificity in DNA binding of ABI3s was also demonstrated with nuclear extracts of cultured cells of Arachis hypogea. Supershift analysis of ABRE and AuxRE bound nuclear proteins with antibodies raised against B2B3 domains of PvAlf revealed that ABI3 associated complexes were detectable in association with both cis elements. Competition GMSA confirmed the same complexes to bind ABRE and AuxRE. This dual specificity of ABI3 like factors in DNA binding targeted to natural promoters responsive to ABA and auxin suggests them to have a potential role in conferring crosstalk between these two phytohormones.
