Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates.

DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wild-type cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.

PCNA and Msh2-Msh6 activate an Mlh1-Pms1 endonuclease pathway required for Exo1-independent mismatch repair.

Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-Pms1 recruitment, including Exonuclease 1 (Exo1)-dependent and -independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-Pms1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyperaccumulation of repair intermediate Mlh1-Pms1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-Pms1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System.

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins.

A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispair-containing DNA substrates. A mixture of MutS homolog 2 (Msh2)-MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5' or 3' strand interruption with different efficiencies. The Msh2-MutS homolog 3 mispair recognition protein could substitute for the Msh2-Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1-postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5' or 3' strand interruption occurred by mispair binding-dependent 5' excision and subsequent resynthesis with excision tracts of up to ~2.9 kb occurring during the repair of the substrate with a 3' strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Template switching during break-induced replication.

DNA double-strand breaks (DSBs) are potentially lethal lesions that arise spontaneously during normal cellular metabolism, as a consequence of environmental genotoxins or radiation, or during programmed recombination processes. Repair of DSBs by homologous recombination generally occurs by gene conversion resulting from transfer of information from an intact donor duplex to both ends of the break site of the broken chromosome. In mitotic cells, gene conversion is rarely associated with reciprocal exchange and thus limits loss of heterozygosity for markers downstream of the site of repair and restricts potentially deleterious chromosome rearrangements. DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). BIR from one of the two ends of a DSB would result in loss of heterozygosity, suggesting that BIR is suppressed when DSBs have two ends so that repair occurs by the more conservative gene conversion mechanism. Here we show that BIR can occur by several rounds of strand invasion, DNA synthesis and dissociation. We further show that chromosome rearrangements can occur during BIR if dissociation and reinvasion occur within dispersed repeated sequences. This dynamic process could function to promote gene conversion by capture of the displaced invading strand at two-ended DSBs to prevent BIR.

Explosive Pleistocene diversification and hemispheric expansion of a "great speciator".

Factors that influence speciation rates among groups of organisms are integral to deciphering macroevolutionary processes; however, they remain poorly understood. Here, we use molecular phylogenetic data and divergence time estimates to reconstruct the pattern and tempo of speciation within a widespread and homogeneous bird family (white-eyes, Zosteropidae) that contains an archetypal "great speciator." Our analyses show that the majority of this species-rich family constitutes a clade that arose within the last 2 million years, yielding a per-lineage diversification rate among the highest reported for vertebrates (1.95-2.63 species per million years). However, unlike most rapid radiations reported to date, this burst of diversification was not limited in geographic scope, but instead spanned the entire Old World tropics, parts of temperate Asia, and numerous Atlantic, Pacific, and Indian Ocean archipelagos. The tempo and geographic breadth of this rapid radiation defy any single diversification paradigm, but implicate a prominent role for lineage-specific life-history traits (such as rapid evolutionary shifts in dispersal ability) that enabled white-eyes to respond rapidly and persistently to the geographic drivers of diversification.

A Study of the Feasibility of FDG-PET/CT to Systematically Detect and Quantify Differential Metabolic Effects of Chronic Tobacco Use in Organs of the Whole Body-A Prospective Pilot Study.

RATIONALE AND OBJECTIVES: The aim of this study was to assess the feasibility of 18F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography (PET/CT) to systematically detect and quantify differential effects of chronic tobacco use in organs of the whole body. MATERIALS AND METHODS: Twenty healthy male subjects (10 nonsmokers and 10 chronic heavy smokers) were enrolled. Subjects underwent whole-body FDG-PET/CT, diagnostic unenhanced chest CT, mini-mental state examination, urine testing for oxidative stress, and serum testing. The organs of interest (thyroid, skin, skeletal muscle, aorta, heart, lung, adipose tissue, liver, spleen, brain, lumbar spinal bone marrow, and testis) were analyzed on FDG-PET/CT images to determine their metabolic activities using standardized uptake value (SUV) or metabolic volumetric product (MVP). Measurements were compared between subject groups using two-sample t tests or Wilcoxon rank-sum tests as determined by tests for normality. Correlational analyses were also performed. RESULTS: FDG-PET/CT revealed significantly decreased metabolic activity of lumbar spinal bone marrow (MVPmean: 29.8 ± 9.7 cc vs 40.8 ± 11.6 cc, P = 0.03) and liver (SUVmean: 1.8 ± 0.2 vs 2.0 ± 0.2, P = 0.049) and increased metabolic activity of visceral adipose tissue (SUVmean: 0.35 ± 0.10 vs 0.26 ± 0.06, P = 0.02) in chronic smokers compared to nonsmokers. Normalized visceral adipose tissue volume was also significantly decreased (P = 0.04) in chronic smokers. There were no statistically significant differences in the metabolic activity of other assessed organs. CONCLUSIONS: Subclinical organ effects of chronic tobacco use are detectable and quantifiable on FDG-PET/CT. FDG-PET/CT may, therefore, play a major role in the study of systemic toxic effects of tobacco use in organs of the whole body for clinical or research purposes.

Aberrant double-strand break repair resulting in half crossovers in mutants defective for Rad51 or the DNA polymerase delta complex.

Homologous recombination is an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Most DSB repair events occur by gene conversion limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and restricting deleterious chromosome rearrangements. DSBs with only one end available for repair undergo strand invasion into a homologous duplex DNA, followed by replication to the chromosome end (break-induced replication [BIR]), leading to LOH for all markers downstream of the site of strand invasion. Using a transformation-based assay system, we show that most of the apparent BIR events that arise in diploid Saccharomyces cerevisiae rad51Delta mutants are due to half crossovers instead of BIR. These events lead to extensive LOH because one arm of chromosome III is deleted. This outcome is also observed in pol32Delta and pol3-ct mutants, defective for components of the DNA polymerase delta (Pol delta) complex. The half crossovers formed in Pol delta complex mutants show evidence of limited homology-dependent DNA synthesis and are partially Mus81 dependent, suggesting that strand invasion occurs and the stalled intermediate is subsequently cleaved. In contrast to rad51Delta mutants, the Pol delta complex mutants are proficient for repair of a 238-bp gap by gene conversion. Thus, the BIR defect observed for rad51 mutants is due to strand invasion failure, whereas the Pol delta complex mutants are proficient for strand invasion but unable to complete extensive tracts of recombination-initiated DNA synthesis.

Break-induced replication: what is it and what is it for?

Homologous recombination (HR) is considered to be an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Indeed, most DSB repair events occur by a non-crossover mechanism limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and preventing chromosome rearrangements. However, DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). As BIR from one of the two ends of a DSB would result in a long tract of LOH it suggests BIR is suppressed when DSBs have two ends in order for repair to occur by a more conservative HR mechanism. Recent studies showed that BIR can occur by several rounds of strand invasion, DNA synthesis and dissociation resulting in chromosome rearrangements when dissociation and reinvasion occur within dispersed repeated sequences. Thus template switching BIR can be highly mutagenic and this process could be important for genome evolution and disease development.

Molecular phylogenetics of monarch flycatchers (genus Monarcha) with emphasis on Solomon Island endemics.

Systematic relationships among monarch flycatchers (genus Monarcha) are poorly understood despite dramatic patterns of morphological differentiation that have long attracted the attention of evolutionary biologists. With sequence data from the mitochondrial ND2 gene and Control Region, we produced a phylogenetic hypothesis for evolutionary relationships within Monarcha and among the biogeographically complex Solomon Island endemics. Outgroup analyses contradicted monophyly of the genus by imbedding a representative of the genus Clytorhynchus within one of two major clades recovered within Monarcha. These two monarch clades generally correspond with ecological and morphological distinctions, suggesting the genus may warrant revision pending the inclusion of taxa currently allied with Clytorhynchus (e.g., Neolalage spp.). Maximum likelihood reconstructions support monophyletic groupings of the two endemic Solomon Island monarch radiations, however, two currently recognized superspecies (Monarcha manadensis and M. melanopsis) were polyphyletic and paraphyletic, respectively. Interestingly, molecular and morphological differentiation were strikingly decoupled among several Solomon Island endemics and between migratory and non-migratory forms of Monarcha trivirgatus in eastern Australia, suggesting morphological evolution has masked the true history of speciation in these groups. This initial phylogeny provides a novel platform for ongoing exploration of the history underlying dramatic patterns of geographic variation among tropical Pacific flycatchers.