Multivalent binding of the tardigrade Dsup protein to chromatin promotes yeast survival and longevity upon exposure to oxidative damage.

Tardigrades are remarkable in their ability to survive extreme environments. The damage suppressor (Dsup) protein is thought responsible for their extreme resistance to reactive oxygen species (ROS) generated by irradiation. Here we show that expression of Ramazzottius varieornatus Dsup in Saccharomyces cerevisiae reduces oxidative DNA damage and extends the lifespan of budding yeast exposed to chronic oxidative genotoxicity. This protection from ROS requires either the Dsup HMGN-like domain or sequences C-terminal to same. Dsup associates with no apparent bias across the yeast genome, using multiple modes of nucleosome binding; the HMGN-like region interacts with both the H2A/H2B acidic patch and H3/H4 histone tails, while the C-terminal region binds DNA. These findings give precedent for engineering an organism by physically shielding its genome to promote survival and longevity in the face of oxidative damage.

Chaperoning histones at the DNA repair dance.

Unlike all other biological molecules that are degraded and replaced if damaged, DNA must be repaired as chromosomes cannot be replaced. Indeed, DNA endures a wide variety of structural damage that need to be repaired accurately to maintain genomic stability and proper functioning of cells and to prevent mutation leading to disease. Given that the genome is packaged into chromatin within eukaryotic cells, it has become increasingly evident that the chromatin context of DNA both facilitates and regulates DNA repair processes. In this review, we discuss mechanisms involved in removal of histones (chromatin disassembly) from around DNA lesions, by histone chaperones and chromatin remodelers, that promotes accessibility of the DNA repair machinery. We also elaborate on how the deposition of core histones and specific histone variants onto DNA (chromatin assembly) during DNA repair promotes repair processes, the role of histone post translational modifications in these processes and how chromatin structure is reestablished after DNA repair is complete.

Chromatin Modifiers Alter Recombination Between Divergent DNA Sequences.

Recombination between divergent DNA sequences is actively prevented by heteroduplex rejection mechanisms. In baker's yeast, such antirecombination mechanisms can be initiated by the recognition of DNA mismatches in heteroduplex DNA by MSH proteins, followed by recruitment of the Sgs1-Top3-Rmi1 helicase-topoisomerase complex to unwind the recombination intermediate. We previously showed that the repair/rejection decision during single-strand annealing recombination is temporally regulated by MSH (MutShomolog) protein levels and by factors that excise nonhomologous single-stranded tails. These observations, coupled with recent studies indicating that mismatch repair (MMR) factors interact with components of the histone chaperone machinery, encouraged us to explore roles for epigenetic factors and chromatin conformation in regulating the decision to reject vs. repair recombination between divergent DNA substrates. This work involved the use of an inverted repeat recombination assay thought to measure sister chromatid repair during DNA replication. Our observations are consistent with the histone chaperones CAF-1 and Rtt106, and the histone deacetylase Sir2, acting to suppress heteroduplex rejection and the Rpd3, Hst3, and Hst4 deacetylases acting to promote heteroduplex rejection. These observations, and double-mutant analysis, have led to a model in which nucleosomes located at DNA lesions stabilize recombination intermediates and compete with MMR factors that mediate heteroduplex rejection.

Genomic Instability Promoted by Overexpression of Mismatch Repair Factors in Yeast: A Model for Understanding Cancer Progression.

Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a variety of cancers showed that increased expression of MMR proteins often correlated with tumor aggressiveness, metastasis, and early recurrence. To better understand these observations, we used The Cancer Genome Atlas and Gene Expression across Normal and Tumor tissue databases to analyze MMR protein expression in cancers. We found that the MMR genes MSH2 and MSH6 are overexpressed more frequently than MSH3, and that MSH2 and MSH6 are often cooverexpressed as a result of copy number amplifications of these genes. These observations encouraged us to test the effects of upregulating MMR protein levels in baker's yeast, where we can sensitively monitor genome instability phenotypes associated with cancer initiation and progression. Msh6 overexpression (two- to fourfold) almost completely disrupted mechanisms that prevent recombination between divergent DNA sequences by interacting with the DNA polymerase processivity clamp PCNA and by sequestering the Sgs1 helicase. Importantly, cooverexpression of Msh2 and Msh6 (∼eightfold) conferred, in a PCNA interaction-dependent manner, several genome instability phenotypes including increased mutation rate, increased sensitivity to the DNA replication inhibitor HU and the DNA-damaging agents MMS and 4-nitroquinoline N-oxide, and elevated loss-of-heterozygosity. Msh2 and Msh6 cooverexpression also altered the cell cycle distribution of exponentially growing cells, resulting in an increased fraction of unbudded cells, consistent with a larger percentage of cells in G1. These novel observations suggested that overexpression of MSH factors affected the integrity of the DNA replication fork, causing genome instability phenotypes that could be important for promoting cancer progression.

Understanding how mismatch repair proteins participate in the repair/anti-recombination decision.

Mismatch repair (MMR) systems correct DNA mismatches that result from DNA polymerase misincorporation errors. Mismatches also appear in heteroduplex DNA intermediates formed during recombination between nearly identical sequences, and can be corrected by MMR or removed through an unwinding mechanism, known as anti-recombination or heteroduplex rejection. We review studies, primarily in baker's yeast, which support how specific factors can regulate the MMR/anti-recombination decision. Based on recent advances, we present models for how DNA structure, relative amounts of key repair proteins, the timely localization of repair proteins to DNA substrates and epigenetic marks can modulate this critical decision.

A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences in Saccharomyces cerevisiae.

Single-strand annealing (SSA) is an important homologous recombination mechanism that repairs DNA double strand breaks (DSBs) occurring between closely spaced repeat sequences. During SSA, the DSB is acted upon by exonucleases to reveal complementary sequences that anneal and are then repaired through tail clipping, DNA synthesis, and ligation steps. In baker's yeast, the Msh DNA mismatch recognition complex and the Sgs1 helicase act to suppress SSA between divergent sequences by binding to mismatches present in heteroduplex DNA intermediates and triggering a DNA unwinding mechanism known as heteroduplex rejection. Using baker's yeast as a model, we have identified new factors and regulatory steps in heteroduplex rejection during SSA. First we showed that Top3-Rmi1, a topoisomerase complex that interacts with Sgs1, is required for heteroduplex rejection. Second, we found that the replication processivity clamp proliferating cell nuclear antigen (PCNA) is dispensable for heteroduplex rejection, but is important for repairing mismatches formed during SSA. Third, we showed that modest overexpression of Msh6 results in a significant increase in heteroduplex rejection; this increase is due to a compromise in Msh2-Msh3 function required for the clipping of 3' tails. Thus 3' tail clipping during SSA is a critical regulatory step in the repair vs. rejection decision; rejection is favored before the 3' tails are clipped. Unexpectedly, Msh6 overexpression, through interactions with PCNA, disrupted heteroduplex rejection between divergent sequences in another recombination substrate. These observations illustrate the delicate balance that exists between repair and replication factors to optimize genome stability.