Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase

The Bloom's syndrome helicase, BLM, is a member of the highly conserved RecQ family, and possesses both DNA unwinding and DNA strand annealing activities. BLM also promotes branch migration of Holliday junctions. One role for BLM is to act in conjunction with topoisomerase IIIα to process homologous recombination (HR) intermediates containing a double Holliday junction by a process termed dissolution. However, several lines of evidence suggest that BLM may also act early in one or more of the recombination pathways to eliminate illegitimate or aberrantly paired DNA joint molecules. We have investigated whether BLM can disrupt DNA displacement loops (D-loops), which represent the initial strand invasion step of HR. We show that mobile D-loops created by the RecA recombinase are a highly preferred substrate for BLM with the invading strand being displaced from the duplex. We have identified structural features of the D-loop that determine the efficiency with which BLM promotes D-loop dissociation. We discuss these results in the context of models for the role of BLM as an ‘anti-recombinase’

The Roles of the Saccharomyces cerevisiae RecQ Helicase SGS1 in Meiotic Genome Surveillance

leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids. chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout meiosis

Dependence on RAD52 and RAD1 for anticancer drug resistance mediated by inactivation of mismatch repair genes

AbstractMismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3,4]. We report that inactivation of MMR genes (MLH1, MLH2, MSH2, MSH3, MSH6, but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1, MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins – in addition to their role in DNA-damage recognition – decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression

Temperature-Dependent Modulation of Chromosome Segregation in msh4 Mutants of Budding Yeast

BACKGROUND:In many organisms, homologous chromosomes rely upon recombination-mediated linkages, termed crossovers, to promote their accurate segregation at meiosis I. In budding yeast, the evolutionarily conserved mismatch-repair paralogues, Msh4 and Msh5, promote crossover formation in conjunction with several other proteins, collectively termed the Synapsis Initiation Complex (SIC) proteins or 'ZMM's (Zip1-Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3). zmm mutants show decreased levels of crossovers and increased chromosome missegregation, which is thought to cause decreased spore viability. PRINCIPAL FINDINGS:In contrast to other ZMM mutants, msh4 and msh5 mutants show improved spore viability and chromosome segregation in response to elevated temperature (23 degrees C versus 33 degrees C). Crossover frequencies in the population of viable spores in msh4 and msh5 mutants are similar at both temperatures, suggesting that temperature-mediated chromosome segregation does not occur by increasing crossover frequencies. Furthermore, meiotic progression defects at elevated temperature do not select for a subpopulation of cells with improved segregation. Instead, another ZMM protein, Zip1, is important for the temperature-dependent improvement in spore viability. CONCLUSIONS:Our data demonstrate interactions between genetic (zmm status) and environmental factors in determining chromosome segregation

The new yeast is a mouse

Recombination hotspots are determined not only by features of the local genome but also by sequences acting at a considerable distance both incis andtrans

The function of exonuclease I in meiotic recombination : a genetic and physical analysis

Exo1 is a member of the Rad2 protein family and possesses both 5’-3’ exonuclease and 5’ flap endonuclease activities. In addition to performing a variety of functions during mitotic growth, Exo1 is also important for the production of crossovers during meiosis. However, its precise molecular role has remained ambiguous and several models have been proposed to account for the crossover deficit observed in its absence. Here, physical evidence that the nuclease activity of Exo1 is essential for normal 5’-3’ resection at the Spo11-dependent HIS4 hotspot in otherwise wild-type cells is presented. This same activity was also required for normal levels of gene conversion at the locus. Nevertheless, gene conversions were frequently observed at a distance beyond that at which resection was readily detectable arguing that it is not the extent of the initial DNA end resection that limits heteroduplex formation. In addition to these nuclease-dependent functions, nuclease-deficient exo1 mutants were found to be capable of maintaining crossing-over at wild-type levels in a number of genetic intervals, suggesting that Exo1 also plays a nuclease-independent role in crossover promotion. Furthermore, the results of both physical and genetic analyses imply that Sgs1 does not contribute significantly to resection during meiosis in exo1∆ cells, indicating that the mitotic and meiotic resection machinery differs. In light of these new insights, a model describing the formation of heteroduplex DNA and crossovers during meiosis is proposed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Multiple mechanisms mediating the starvation induced activation of recombination at HIS4 in Saccharomyces cerevisiae

Meiotic recombination occurs at relatively high levels at specific regions in the genome called hotspots. The transcription factor-dependent hotspots (α-hotspots) have been widely studied in yeast, and are beginning to be elucidated in mammals. The HIS4 hotspot activity in Saccharomyces cerevisiae requires binding of Bas1p, Bas2p, Rap1p and Gcn4p. Bas1p acts in conjunction with Bas2p to regulate basal level of transcription of their target genes, and can be stimulated under conditions of adenine starvation and accumulation of metabolites AICAR and SAICAR from the purine biosynthesis pathway. Gcn4p activates transcription of yeast genes in response to starvation for amino acids and purines. This study focused on the influence of nutritional starvation on HIS4 hotspot activity, and different mechanisms mediating this effect. Our data suggests that deletion of genes known to accumulate AICAR/SAICAR can stimulate recombination at HIS4 in a Bas1p-dependent manner. Furthermore, intracellular and extracellular starvation for adenine and amino acids also activates recombination at HIS4. In addition, moderate levels of starvation only affect recombination when chromatin is already hyperacetylated, by the inactivation of the Set2p methyltransferase. Bas1p plays an essential role in mediating the effect of starvation and the set2 mutation on recombination. We showed that Gcn4p is not required for HIS4 hotspot activity, but plays a modest role in the effect of starvation in an adenine auxotrophic strain. Additionally, the starvation effect is also mediated by an as yet unknown factor independent from Bas1p/Bas2p and Gcn4p. This work provides additional information regarding the regulation of a transcription factor-dependent hotspot activity, and factors influencing its activation. Furthermore, data in this study indicate that BAS1, and not BAS2 exhibit haploinsufficiency with respect to its function in activating meiotic recombination. This implies that Bas1p is rate-limiting for HIS4 hotspot activity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo