DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination.

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Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

Subject Collection DNA Recombination Models of Recombinational DNA RepairSources of DNA Double-Strand Breaks andAnuja Mehta and James E. HaberMeets DNATranscription and Recombination: When RNAAndres Aguilera and Helene GaillardMechanism and RegulationEnd Resection at Double-Strand Breaks:Lorraine S. SymingtonResection in Homologous RecombinationStructural Studies of DNA End Detection andChristian Linke-Winnebeck, et al.Christian Bernd Schiller, Florian Ulrich Seifert,The Dissolution of Double Holliday JunctionsAnna H. Bizard and Ian D. HicksonFor additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/ Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reservedDownloaded from http://cshperspectives.cshlp.org/

Sources of DNA Double-Strand Breaks and Models of Recombinational

Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATα. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATα1, and MATα2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break.

https://www.genetics.org/content/genetics/191/1/33.full.pdf

Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae

N-terminal methionine excision (NME) is the major proteolytic pathway responsible for the diversity of N-terminal amino acids in proteins. Dedicated NME components have been identified in all organisms, in all compartments in which protein synthesis occurs: cytoplasm, plastids and mitochondria. Recent studies have revealed that NME is regulated at various levels and plays an important role in controlling protein turnover. NME is essential in Eubacteria and lower eukaryotes and is the target of many natural and synthetic inhibitors. Such inhibitors have considerable potential for use in the treatment of various human diseases, from cancer to bacterial and parasitic infections.

Protein N-terminal methionine excision.

Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell.

https://www.genetics.org/content/genetics/198/3/795.full.pdf

Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

A survey of polypeptide deformylase function throughout the eubacterial lineage.

During Saccharomyces cerevisiae mating-type switching, an HO endonuclease-induced double-strand break (DSB) at MAT is repaired by recombining with one of two donors, HMLα or HMRa, located at opposite ends of chromosome III. MATa cells preferentially recombine with HMLα; this decision depends on the Recombination Enhancer (RE), located about 17 kb to the right of HML. In MATα cells, HML is rarely used and RE is bound by the MATα2-Mcm1 corepressor, which prevents the binding of other proteins to RE. In contrast, in MATa cells, RE is bound by multiple copies of Fkh1 and a single copy of Swi4/Swi6. We report here that, when RE is replaced with four LexA operators in MATa cells, 95% of cells use HMR for repair, but expression of a LexA-Fkh1 fusion protein strongly increases HML usage. A LexA-Fkh1 truncation, containing only Fkh1's phosphothreonine-binding FHA domain, restores HML usage to 90%. A LexA-FHA-R80A mutant lacking phosphothreonine binding fails to increase HML usage. The LexA-FHA fusion protein associates with chromatin in a 10-kb interval surrounding the HO cleavage site at MAT, but only after DSB induction. This association occurs even in a donorless strain lacking HML. We propose that the FHA domain of Fkh1 regulates donor preference by physically interacting with phosphorylated threonine residues created on proteins bound near the DSB, thus positioning HML close to the DSB at MAT. Donor preference is independent of Mec1/ATR and Tel1/ATM checkpoint protein kinases but partially depends on casein kinase II. RE stimulates the strand invasion step of interchromosomal recombination even for non-MAT sequences. We also find that when RE binds to the region near the DSB at MATa then Mec1 and Tel1 checkpoint kinases are not only able to phosphorylate histone H2A (γ-H2AX) around the DSB but can also promote γ-H2AX spreading around the RE region.

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Regulation of budding yeast mating-type switching donor preference by the FHA domain of Fkh1.

Saccharomyces mating-type switching results from replacement by gene conversion of the MAT locus with sequences copied from one of two unexpressed donor loci, HML or HMR. MATa cells recombine with HMLalpha approximately 90% of the time, whereas MATalpha cells choose HMRa 80%-90% of the time. HML preference in MATa is controlled by the cis-acting recombination enhancer (RE) that regulates recombination along the entire left arm of chromosome III. Comparison of RE sequences between S. cerevisiae, S. carlsbergensis, and S. bayanus defines four highly conserved regions (A, B, C, and D) within a 270-bp minimum RE. An adjacent E region enhances RE activity. Multimers of region A, D, or E are sufficient to promote selective use of HML. Regions A, D, and E each bind in vivo the transcription activator forkhead proteins Fkh1p and Fkh2p and their associated Ndd1p, although there are no adjacent open reading frames (ORFs). Deletion of FKH1 significantly reduces MATa's use of HML, as does mutation of the Fkh1/Fkh2-binding sites in a multimer of region A. We conclude that Fkh1p regulates MATa donor preference through direct interaction with RE.

/pdf/saccharomyces-forkhead-protein-fkh1-regulates-donor-5el0b01c75.pdf

Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer.

In wild-type diploid cells, heteroallelic recombination between his4A and his4C alleles leads mostly to His + gene conversions that have a parental configuration of flanking markers, but ∼22% of recombinants have associated reciprocal crossovers In rad52 strains, gene conversion is reduced 75-fold and the majority of His + recombinants are crossover associated, with the largest class being half-crossovers in which the other participating chromatid is lost We report that UV irradiating rad52 cells results in an increase in overall recombination frequency, comparable to increases induced in wild-type (WT) cells, and surprisingly results in a pattern of recombination products quite similar to RAD52 cells: gene conversion without exchange is favored, and the number of 2 n − 1 events is markedly reduced Both spontaneous and UV-induced RAD52 -independent recombination depends strongly on Rad50, whereas rad50 has no effect in cells restored to RAD52  The high level of noncrossover gene conversion outcomes in UV-induced rad52 cells depends on Rad51, but not on Rad59 Those outcomes also rely on the UV-inducible kinase Dun1 and Dun19s target, the repressor Crt1, whereas gene conversion events arising spontaneously depend on Rad59 and Crt1 Thus, there are at least two Rad52-independent recombination pathways in budding yeast

https://www.genetics.org/content/genetics/179/1/199.full.pdf

Mechanisms of Rad52-Independent Spontaneous and UV-Induced Mitotic Recombination in Saccharomyces cerevisiae

Recombination events between non-identical sequences most often involve heteroduplex DNA intermediates that are subjected to mismatch repair. The well-characterized long-patch mismatch repair process, controlled in eukaryotes by bacterial MutS and MutL orthologs, is the major system involved in repair of mispaired bases. Here we present evidence for an alternative short-patch mismatch repair pathway that operates on a broad spectrum of mismatches. In msh2 mutants lacking the long-patch repair system, sequence analysis of recombination tracts resulting from exchanges between similar but non-identical (homeologous) parental DNAs showed the occurrence of short-patch repair events that can involve <12 nucleotides. Such events were detected both in mitotic and in meiotic recombinants. Confirming the existence of a distinct short-patch repair activity, we found in a recombination assay involving homologous alleles that closely spaced mismatches are repaired independently with high efficiency in cells lacking MSH2 or PMS1. We show that this activity does not depend on genes required for nucleotide excision repair and thus differs from the short-patch mismatch repair described in Schizosaccharomyces pombe.

/pdf/evidence-for-short-patch-mismatch-repair-in-saccharomyces-1suf73hbn9.pdf

Eric Coïc

Papers

A survey of polypeptide deformylase function throughout the eubacterial lineage.

Regulation of budding yeast mating-type switching donor preference by the FHA domain of Fkh1.

Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer.

Mechanisms of Rad52-Independent Spontaneous and UV-Induced Mitotic Recombination in Saccharomyces cerevisiae

Evidence for short‐patch mismatch repair in Saccharomyces cerevisiae