The role of Arp4 and histone H4 acetylation in DNA repair and genome stability

All cellular processes involving DNA and chromosomes must take place in the context of chromatin. It is becoming increasingly clear that chromatin remodeling complexes play very important roles in these processes, and that various post-translational modifications on histone proteins are critical for their proper regulation. In this dissertation I show that acetylation of histone H4 is required for DNA repair of double-strand breaks (DSBs). This repair function also requires Arp4, a component of the NuA4 histone acetyltransferase (HAT) complex. Mutations in Arp4 and histone H4 which result in diminished H4 acetylation also result in hypersensitivity to DNA damage and defects in non-homologous end joining (NHEJ). Furthermore, I show that Arp4 is specifically recruited to DNA DSBs generated in vivo. These data provide evidence that the NuA4 complex is required for acetylation of histone H4 and participates directly in DNA repair.; The Arp4 protein has additional roles beyond DNA repair. It is also essential for cell viability, and required for transcription of specific genes. Arp4 is found not only in NuA4, but also in at least one other chromatin remodeling complex, INO80. Both of these complexes have been implicated in DNA repair (Shen et al. 2000; Bird et al. 2002), although their chromatin remodeling mechanisms are very different (HAT activity vs. DNA-dependent ATPase activity). In addition, both complexes have been shown to be involved in transcription and are also suggested to be important for other aspects of DNA metabolism such as replication (Steger et al. 1998; Allard et al. 1999; Choy and Kron 2002). I have isolated conditional mutants of ARP4 to study its roles in the cell.{09}In addition to its role in DNA repair, I have found that Arp4 has an essential role related to cell-cycle progression through the G1/S transition, defining a previously unrecognized role for this protein and for chromatin remodeling complexes.; Lastly, I have investigated genetically the mechanism of Sir2 activity in silencing. Sir2 is a NAD-dependent protein deacetylase with conserved homologs from bacteria to humans, and is required for heterochromatin formation and gene silencing in yeast at HM sites, telomeres and the rDNA locus. It has previously been reported that Sir2 deacetylates lysine 16 of H4 and lysines 9 and 14 of H3 in vitro (lmai et al. 2000), and genetic studies of mutations of these lysines suggest they are required for silencing (Johnson et al. 1990; Megee et al. 1990; Park and Szostak 1990; Hecht et al. 1995; Braunstein et al. 1996). However, to date there has been no direct evidence that the primary role of Sir2 in gene silencing in vivo is actually deacetylation of these lysines. I have found genetic evidence that the essential function of Sir2 in silencing can not be explained simply by the deacetylation of H4 Lys16 and H3 Lys9 and Lys14. This suggests that a previously unrecognized enzymatic activity or substrate of Sir2 is required for silencing.