Although Hat1p has long been presumed to be involved in chromatin assembly through the acetylation of newly synthesized histone H4 on lysine 5 and 12, there has been lack of straightforward evidence linking Hat1p to this process. We utilized the yeast Gal-HO system to study the function of nuclear Hat1p-containing type B histone acetyltransferase complex (NuB4) in DNA double strand break repair linked chromatin assembly. We found that each of the NuB4 components affects repair-linked chromatin reassembly but that their contributions are not equivalent. In particular, deletion of the catalytic subunit, Hat1p, caused a significant defect in chromatin reassembly. In addition, loss of the histone chaperone Hif1p, when combined with an allele of H3 that mutates lysines 14 and 23 to arginine, has a pronounced defect in chromatin reassembly that is similar to that observed in an asf1Δ. Lastly, the role of Hat1p and Hif1p is at least partially redundant with the role of the histone chaperone Asf1p in the DNA repair linked chromatin reassembly process.
The acetylation on lysine 5 and 12 of newly synthesized histone H4 is highly conserved and is temporally correlated with the process of chromatin assembly. However, this pattern of modification has not been shown to be essential for either cell viability or chromatin assembly in any model organism. Using genetic assays in S. cerevisiae, we demonstrated that mutations in histone H4 lysines 5 and 12 confer hypersensitivity to replication stress and DNA damaging agents when combined with mutations in histone H4 lysine 91, which has also been found to be a site of acetylation in the core domain of soluble histone H4. We also showed that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double strand breaks in yeast cells. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation are also defective in DNA damage response signaling as the mutants show a marked decrease in the levels of phosphorylated H2A on chromatin 20 kb from double strand break. This decrease is not the result of an inability to generate phosphorylated histone H2A but to an inability to localize this modified histone to chromatin. Therefore, acetylation on newly synthesized histone H4 is required for proper chromatin assembly and maintenance of chromatin structure.
Using the technology called iPOND (isolation of proteins on nascent DNA), we were able to show direct biochemical evidence that mammalian Hat1p is involved in DNA replication coupled chromatin assembly. We found that mouse Hat1p influences newly synthesized histone H4 lysine 5 and 12 acetylation levels in the cell as well as on nascent chromatin. Also, we showed that in the absence of Hat1p, acetylation pattern of histone H3 NH2-terminal tail is different from wild type cells. Finally, we provided evidence that mouse Hat1p is associated with replication forks.
|Commitee:||Herman, Paul, Hopper, James, Ma, Jiyan|
|School:||The Ohio State University|
|Department:||Molecular, Cellular and Developmental Biology|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Keywords:||Chromatin assembly, Dna damage repair, Histone acetylation|
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