Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.

Budding yeast Slx4 interacts with the structure-specific endonuclease Slx1 to ensure completion of ribosomal DNA replication. Slx4 also interacts with the Rad1-Rad10 endonuclease to control cleavage of 3' flaps during repair of double-strand breaks (DSBs). Here we describe the identification of human SLX4, a scaffold for DNA repair nucleases XPF-ERCC1, MUS81-EME1, and SLX1. SLX4 immunoprecipitates show SLX1-dependent nuclease activity toward Holliday junctions and MUS81-dependent activity toward other branched DNA structures. Furthermore, SLX4 enhances the nuclease activity of SLX1, MUS81, and XPF. Consistent with a role in processing recombination intermediates, cells depleted of SLX4 are hypersensitive to genotoxins that cause DSBs and show defects in the resolution of interstrand crosslink-induced DSBs. Depletion of SLX4 causes a decrease in DSB-induced homologous recombination. These data show that SLX4 is a regulator of structure-specific nucleases and that SLX4 and SLX1 are important regulators of genome stability in human cells.

Phosphorylation of Slx4 by Mec1 and Tel1 regulates the single-strand annealing mode of DNA repair in budding yeast.

Budding yeast (Saccharomyces cerevisiae) Slx4 is essential for cell viability in the absence of the Sgs1 helicase and for recovery from DNA damage. Here we report that cells lacking Slx4 have difficulties in completing DNA synthesis during recovery from replisome stalling induced by the DNA alkylating agent methyl methanesulfonate (MMS). Although DNA synthesis restarts during recovery, cells are left with unreplicated gaps in the genome despite an increase in translesion synthesis. In this light, epistasis experiments show that SLX4 interacts with genes involved in error-free bypass of DNA lesions. Slx4 associates physically, in a mutually exclusive manner, with two structure-specific endonucleases, Rad1 and Slx1, but neither of these enzymes is required for Slx4 to promote resistance to MMS. However, Rad1-dependent DNA repair by single-strand annealing (SSA) requires Slx4. Strikingly, phosphorylation of Slx4 by the Mec1 and Tel1 kinases appears to be essential for SSA but not for cell viability in the absence of Sgs1 or for cellular resistance to MMS. These results indicate that Slx4 has multiple functions in responding to DNA damage and that a subset of these are regulated by Mec1/Tel1-dependent phosphorylation.

DNA repair: the importance of phosphorylating histone H2AX.

How phosphorylated histone H2AX, known as gamma-H2AX, functions in the cellular response to DNA double-strand breaks is the subject of intensive investigation. Recent research in yeast and mammalian cells shows that gamma-H2AX facilitates post-replicational DNA repair by recruiting cohesin, a protein complex that holds sister chromatids together.

Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation.

In budding yeast, the Rad9 protein is an important player in the maintenance of genomic integrity and has a well-characterised role in DNA damage checkpoint activation. Recently, roles for different post-translational histone modifications in the DNA damage response, including H2A serine 129 phosphorylation and H3 lysine 79 methylation, have also been demonstrated. Here, we show that Rad9 recruitment to foci and bulk chromatin occurs specifically after ionising radiation treatment in G2 cells. This stable recruitment correlates with late stages of double strand break (DSB) repair and, surprisingly, it is the hypophosphorylated form of Rad9 that is retained on chromatin rather than the hyperphosphorylated, checkpoint-associated, form. Stable Rad9 accumulation in foci requires the Mec1 kinase and two independently regulated histone modifications, H2A phosphorylation and Dot1-dependent H3 methylation. In addition, Rad9 is selectively recruited to a subset of Rad52 repair foci. These results, together with the observation that rad9Delta cells are defective in repair of IR breaks in G2, strongly indicate a novel post checkpoint activation role for Rad9 in promoting efficient repair of DNA DSBs by homologous recombination.

Multiple approaches to study S. cerevisiae Rad9, a prototypical checkpoint protein.

The Saccharomyces cerevisiae RAD9 checkpoint gene is the prototypical checkpoint gene and is required for efficient checkpoint regulation in late G1, S, and at the G2/M cell cycle transition following DNA damage. Rad9 is required for the activation of Rad53 after damage and has been proposed to have roles in lesion recognition as well as DNA repair and the maintenance of genome stability. Here we describe methodology suitable for the study of G1, intra-S, and G2/M checkpoints in budding yeast, the analysis of Rad9/Rad53 phospho-forms, the biochemical analysis of Rad9 and Rad53, the fractionation of soluble and chromatin associated proteins, including Rad9, and the live cell imaging of GFP tagged Rad9.

Mec1/Tel1-dependent phosphorylation of Slx4 stimulates Rad1-Rad10-dependent cleavage of non-homologous DNA tails.

Budding yeast Slx4 interacts with the Rad1-Rad10 endonuclease that is involved in nucleotide excision repair (NER), homologous recombination (HR) and single-strand annealing (SSA). We previously showed that Slx4 is dispensable for NER but is essential for SSA. Slx4 is phosphorylated by the Mec1 and Tel1 kinases after DNA damage on at least six Ser/Thr residues, and mutation of all six residues to Ala reduces the efficiency of SSA. In this study, we further investigated the role of Slx4 phosphorylation in SSA, specifically in regulating cleavage of 3' non-homologous (NH) DNA tails by Rad1-Rad10 during SSA and HR. Slx4 became phosphorylated after induction of a single double-strand break (DSB) during SSA and dephosphorylation coincided approximately with completion of repair. Slx4 is recruited to 3' NH tails during DSB repair, but this does not require phosphorylation of Slx4. However, we identified a specific damage-dependent Mec1/Tel1 site of Slx4 phosphorylation, Thr 113, that is required for efficient cleavage of NH tails by Rad1-Rad10. Consistent with these data, deletion of both Mec1 and Tel1 severely reduces the efficiency of NH DNA tail cleavage during HR. These data show that phosphorylation of Slx4 by Mec1 and Tel1 plays an important role in facilitating NH DNA tail cleavage during HR.