The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice

Abstract

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5'-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.

Keywords: DNA double strand break; NHEJ; Saccharomyces cerevisiae; histone acetylation; histone methylation; histone phosphorylation; histone ubiquitylation; homologous recombination.

Figure 1

Mechanisms of DSB repair. (a) Non-homologous end joining (NHEJ) directly rejoins the two broken ends together. (b) Resection generates a 3′-ended ssDNA tail (in dark blue) that invades a homologous duplex (in red) and stimulates DNA synthesis. If the elongating strand re-anneals with the broken template, DSB is repaired by synthesis-dependent strand annealing (SDSA), thus generating only non-crossover products (left). If the intermediate is stabilized by a second-end capture, a double Holliday junction is formed. The resolution of this intermediate generates both crossover and non-crossover products (center). A failure to engage the second DSB end leads to break-induced replication (BIR). The 3′ overhang that has invaded the homologous template triggers bubble migration and extensive DNA synthesis that can proceed until the end of the chromosome (right). (c) A DSB between two homologous sequences (in light blue) can be repaired by single-strand annealing (SSA). When resection generates ssDNA at the homologous sequences, they can anneal to each other. Subsequent ligation causes the deletion of one of the homologous sequences and of the intervening sequence.

Figure 2

Histone modifications and Rad9 recruitment to DSBs in S. cerevisiae. Rad9 is a master regulator of DSB repair pathway choice and its binding to chromatin is regulated by specific histone modifications, comprising H2A phosphorylation by Tel1 and Mec1, H2B ubiquitylation by Bre1-Rad6 complex and H3 methylation by Set1, Set2, and Dot1. Histone PTMs can generate a docking site for Rad9, which binds to phosphorylated H2A S129 residue (γ-H2A) through its BRCT motif and to methylated H3 K79 residue through its Tudor domain. Di- and tri-methylation of H3 K4 and K36 residues likely stabilize the interaction between Rad9 and the chromatin by crosstalk with H2B K123 ubiquitylation. Dotted lines represent molecular interactions with histone marks, dotted lines with arrowheads correspond to epigenetic crosstalk. Created with BioRender.com.

Figure 3

Phosphorylation of histone H2A promotes HR in S. cerevisiae. Tel1- and Mec1-dependent phosphorylation of H2A S129 (γ-H2A) regulates both negatively and positively DNA end resection by recruiting Rad9 and chromatin remodeling enzymes, respectively. γ-H2A also increases chromosome mobility, thus favoring homology search, and promotes cohesin recruitment to the DSB ends. Mec1-dependent phosphorylation of H2A S15 stimulates DNA end resection and promotes HR repair. Created with BioRender.com.

Figure 4

Methylation of histone H3 promotes NHEJ in S. cerevisiae. Set1-dependent tri-methylation of H3 K4 residue stimulates an efficient recruitment of Ku to the DSB ends. Set2-dependent methylation of H3 K36 likely limits DNA end resection and chromatin accessibility. Dot1-dependent methylation of H3 K79 is crucial for Rad9 recruitment, which inhibits DSB end resection. Created with BioRender.com.

Figure 5

Ubiquitylation of histone H2B promotes HR in S. cerevisiae. Bre1-Rad6-dependent ubiquitylation of H2B K123 supports Rad51 loading on resected ends. It also regulates chromatin remodelers like Chd1, thus promoting chromatin accessibility and resection. Finally, H2B K123 ubiquitylation could promote resection by inhibiting Rad9 recruitment and increasing Exo1 association to the DSB ends. Created with BioRender.com.

Figure 6

Histone acetylation promotes HR in S. cerevisiae. Multiple lysine residues of H2A and H4 histones are acetylated by the NuA4 complex, whose recruitment to the DSB ends is supported by the SAGA complex. SAGA, in turn, acetylates multiple H2B and H3 lysine residues. Histone acetylation causes extensive chromatin remodeling and nucleosome eviction, which promotes both DNA end resection and strand invasion. Double arrowheads line represents protein molecular interaction. Created with BioRender.com.