The repair of DNA damage is essential for the prevention of cell death and carcinogenesis. Lethal DNA damage occurs when both strands of DNA are broken, which can lead to the loss of chromosome arms during mitosis (45
). Cells employ two major pathways to repair a double-strand break (DSB), nonhomologous end joining (NHEJ) and homologous recombination. Homologous recombination is mediated by the Rad52 epistasis group of proteins, where Rad52 is the only member essential for homologous recombination (26
). Homologous recombination requires significant sequence homology to allow the lesion to be “patched” by copying the homologous donor sequences. The single defined HO site at the Saccharomyces cerevisiae MAT
locus has allowed the events of DSB repair to be dissected (12
). The HO lesion is primarily repaired by homologous recombination with donor sequences from HML
α or HMRa
). We chose to use the HO system to investigate whether homologous recombination is regulated by chromatin modifications, as is the case for transcriptional regulation.
Chromatin comprises a basic repeating unit called the nucleosome. The nucleosome is made up of a histone octamer (two molecules each of H2A, H2B, H3, and H4) which has approximately 147 base pairs of DNA wrapped around it (25
). There are many types of histone modifications that alter chromatin structure and function, including acetylation, methylation, phosphorylation, ubiquitylation, ADP-ribosylation, and sumoylation. Most of these types of modifications have been found to play a fundamental role in regulating gene expression by modulating the accessibility of the DNA to binding factors and by acting as specific binding sites for factors that regulate transcription (10
). These modifications on histone proteins have been dubbed the histone code (8
Until recently, the evidence for a DNA damage-induced histone code was limited to the phosphorylation of the C-terminal tail of the histone variant H2AX (or H2A in S. cerevisiae
). This is a very early response to double-strand DNA breaks, and recent studies in yeast indicate that H2A phosphorylation is required for the recruitment of chromatin-modifying complexes to the vicinity of DNA damage. Phosphorylated H2A in yeast leads to the recruitment of the chromatin-remodeling complex INO80.com to a double-strand DNA break (32
), which in turn is required for efficient processing of the double-strand break into single-stranded DNA (46
). Similarly, phosphorylation of H2A leads to the recruitment of the Swr1 chromatin-remodeling complex (7
). In addition, phosphorylation of H2A in yeast is required for the recruitment of the NuA4 histone acetyltransferase complex to a region proximal to a double-strand break and is accompanied by localized acetylation of histone H4 on lysine 8 (7
). Methylated and ubiquitinated histones also appear to play an important role in the signaling of double-strand DNA damage to the cell (11
There is a growing body of evidence supporting a role for histone acetylation in double-strand DNA repair. The N terminus of histone H3 has five reversibly acetylatable lysine residues at positions 9, 14, 18, 23, and 27, while the N terminus of histone H4 has four reversibly acetylatable lysine residues at positions 5, 8, 12, and 16. Mutation of all four acetylatable lysines of histone H4 causes sensitivity to double-strand DNA-damaging agents (9
). Mutation of both lysines 14 and 23 on histone H3 increases sensitivity to expression of the HO endonuclease (35
). The enzymes that alter histone acetylation have also been implicated in the repair of DSBs. For example, mutation of a histone acetyltransferase, Gcn5, causes sensitivity to double-strand DNA-damaging agents (6
). A major caveat with these studies linking DNA damage sensitivity to histone acetylation is that it may be a result of indirect effects (for example, transcriptional dysregulation of repair factors). In addition, it is not clear whether the repair is by homologous recombination and/or NHEJ.
There is evidence to suggest that changes in histone acetylation accompany NHEJ, but it is unknown whether histone acetylation changes during homologous recombinational repair. For example, the histone acetyltransferase Esa1 is required for efficient NHEJ (4
). The human counterpart of the Gcn5 histone acetyltransferase is phosphorylated and inactivated by DNA-dependent protein kinase, which is a central component of the NHEJ machinery (2
). There is also clear evidence to suggest that the opposite reaction, histone deacetylation, is important for NHEJ. The histone deacetylase Rpd3 and its binding partner Sin3 are required for efficient NHEJ (18
). Sir3, which is part of a complex containing the histone deacetylase Sir2, is known to relocalize to the site of a DSB in yeast that lacks homologous donor sequences for repair by homologous recombination, but the reason for this is unclear (28
). Similarly, Esa1 localizes to the HO break in yeast that lacks homologous donor sequences for repair by homologous recombination (7
). Two recent reports have shown for the first time that histone acetylation levels actually change around a DSB. Acetylation on H4 K16 is reduced 40 to 50% following induction of a DSB that can be repaired only by NHEJ (18
), and acetylation of lysine H4 K8 transiently increases in chromatin flanking the DSB that can only be repaired by NHEJ (7
). However, we do not know whether histone acetylation changes during homologous recombination.
Since virtually nothing was known about histone acetylation during homologous recombinational repair, we set out to perform a comprehensive analysis of acetylation levels on all nine acetylatable residues of the N-terminal tails of histones H3 and H4 before, during, and after homologous recombinational repair of an HO lesion. We find that localized histone acetylation, followed by histone deacetylation, occurs in a manner dependent on homologous recombinational repair. These modifications are important for viability following DNA repair and are likely to be mediated by the recruitment of the histone acetylases Gcn5 and Esa1 and the histone deacetylases Sir2, Hst1, and Rpd3 during homologous recombination.