Rpd3L and Hda1 histone deacetylases facilitate repair of broken forks by promoting sister chromatid cohesion

Abstract

Genome stability involves accurate replication and DNA repair. Broken replication forks, such as those encountering a nick, lead to double strand breaks (DSBs), which are preferentially repaired by sister-chromatid recombination (SCR). To decipher the role of chromatin in eukaryotic DSB repair, here we analyze a collection of yeast chromatin-modifying mutants using a previously developed system for the molecular analysis of repair of replication-born DSBs by SCR based on a mini-HO site. We confirm the candidates through FLP-based systems based on a mutated version of the FLP flipase that causes nicks on either the leading or lagging DNA strands. We demonstrate that Rpd3L and Hda1 histone deacetylase (HDAC) complexes contribute to the repair of replication-born DSBs by facilitating cohesin loading, with no effect on other types of homology-dependent repair, thus preventing genome instability. We conclude that histone deacetylation favors general sister chromatid cohesion as a necessary step in SCR.

A CDK-regulated chromatin segregase promoting chromosome replication

“…Recombination frequencies were calculated by transforming the indicated yeast strains with the pTINV-FRT plasmid containing an leu2 inverted-repeats recombination system 66 . Recombination tests were performed in mid-log phase cultures after growth at 30°C in liquid SC media with 2 % raffinose and 5 mg/ml doxycycline but lacking uracil.…”

“…Recombination frequencies were calculated by transforming the indicated yeast strains with the pTINV-FRT plasmid containing an leu2 inverted-repeats recombination system 66 .…”

Histone deacetylases facilitate the accurate repair of broken forks

“…Using a powerful-engineered system in which nicks are induced by the action of the yeast endonuclease HO into an incomplete target sequence placed in a reporter gene, we have shown that mutants of the yeast Rpd3 and Hda1 HDACs strongly impair SCR. 2 Furthermore, we have improved the system by substituting the endonuclease HO by a modified version of the flipase (FLP) so that nicks can be specifically targeted to a flipase recognition site located on either DNA strand. This has enabled us to see that the kinetics of repair of broken forks with the sister chromatid is similar regardless of whether the nick was induced in the leading or lagging strand.…”

“…Interestingly, HDACs affect SCR because they are required for the proper loading of cohesins. 2 Cohesin is a conserved multi-subunit protein complex that hold the sister chromatids together thus enabling proper SCR, as shown in yeast and human cells. 4,5 Cohesion is established during replication and lasts until the onset of mitosis to enable the separation of the sister chromatids, but additional cohesins have been shown to load upon DNA damage independently of replication.…”

“…However, Rpd3 and Hda1 loss affect the general loading of cohesins regardless of the induction of DNA damage, supporting that general cohesion, and not only damage-induced cohesion, is crucial for efficient repair by SCR. 2 The cohesion dynamic cycle itself is regulated by acetylation of the cohesin component SMC3 (Structural maintenance of chromosomes protein 3), which is deacetylated by the human HDAC8 class I HDAC, related to yeast Rpd3. However, a deletion mutant in the equivalent yeast HDAC, Hos1 (Hda One Similar 1), which performs this function instead of Rpd3, entails no defect in SCR.…”