283 research outputs found
Serum antioxidants as predictors of the adult respiratory distress syndrome in septic patients
Adult respiratory distress syndrome (ARDS) can develop as a complication of various disorders, including sepsis, but it has not been possible to identify which of the patients at risk will develop this serious disorder. We have investigated the ability of six markers, measured sequentially in blood, to predict development of ARDS in 26 patients with sepsis.
At the initial diagnosis of sepsis (6-24 h before the development of ARDS), serum manganese superoxide dismutase concentration and catalase activity were higher in the 6 patients who subsequently developed ARDS than in 20 patients who did not develop ARDS. These changes in antioxidant enzymes predicted the development of ARDS in septic patients with the same sensitivity, specificity, and efficiency as simultaneous assessments of serum lactate dehydrogenase activity and factor VIII concentration. By contrast, serum glutathione peroxidase activity and Ξ±1Pi-elastase complex concentration did not differ at the initial diagnosis of sepsis between patients who did and did not subsequently develop ARDS, and were not as effective in predicting the development of ARDS.
Measurement of manganese superoxide dismutase and catalase, in addition to the other markers, should facilitate identification of patients at highest risk of ARDS and allow prospective treatment
Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3
double Holliday junction (dHJ) is a central intermediate of homologous recombination that can be processed to yield crossover or non-crossover recombination products. To preserve genomic integrity, cells possess mechanisms to avoid crossing over. We show that Saccharomyces cerevisiae Sgs1 and Top3 proteins are sufficient to migrate and disentangle a dHJ to produce exclusively non-crossover recombination products, in a reaction termed "dissolution." We show that Rmi1 stimulates dHJ dissolution at low Sgs1-Top3 protein concentrations, although it has no effect on the initial rate of Holliday junction (HJ) migration. Rmi1 serves to stimulate DNA decatenation, removing the last linkages between the repaired and template DNA molecules. Dissolution of a dHJ is a highly efficient and concerted alternative to nucleolytic resolution that prevents crossing over of chromosomes during recombinational DNA repair in mitotic cells and thereby contributes to genomic integrity
DNA end resection by Dna2βSgs1βRPA and its stimulation by Top3βRmi1 and Mre11βRad50βXrs2
The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3β²-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5β²-terminated strand of the DNA break and to inhibit 3β² to 5β² degradation by Dna2, actions that generate and protect the 3β²-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11βRad50βXrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3βRmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3βRmi1 and MRX are important for recruitment of the Sgs1βDna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes
Regulatory control of DNA end resection by Sae2 phosphorylation
DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms
Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere Addition at Broken Chromosome Ends
In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5β² to 3β² resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Ξ exo1Ξ strains, where there is little 5β² to 3β² resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Ξ, or exo1Ξ cells. In sgs1Ξ exo1Ξ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5β² to 3β² resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5β² to 3β² resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition
Formation of Complex and Unstable Chromosomal Translocations in Yeast
Genome instability, associated with chromosome breakage syndromes and most human
cancers, is still poorly understood. In the yeast Saccharomyces
cerevisiae, numerous genes with roles in the preservation of genome
integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that
lack Sgs1, a RecQ-like DNA helicase related to the human
Bloom's-syndrome-associated helicase BLM, show an increased rate of
genome instability, and we have previously shown that they accumulate recurring
chromosomal translocations between three similar genes, CAN1,
LYP1 and ALP1. Here, the chromosomal
location, copy number and sequence similarity of the translocation targets
ALP1 and LYP1 were altered to gain insight
into the formation of complex translocations. Among 844 clones with chromosomal
rearrangements, 93 with various types of simple and complex translocations
involving CAN1, LYP1 and ALP1
were identified. Breakpoint sequencing and mapping showed that the formation of
complex translocation types is strictly dependent on the location of the
initiating DNA break and revealed that complex translocations arise via a
combination of interchromosomal translocation and template-switching, as well as
from unstable dicentric intermediates. Template-switching occurred between
sequences on the same chromosome, but was inhibited if the genes were
transferred to different chromosomes. Unstable dicentric translocations
continuously gave rise to clones with multiple translocations in various
combinations, reminiscent of intratumor heterogeneity in human cancers. Base
substitutions and evidence of DNA slippage near rearrangement breakpoints
revealed that translocation formation can be accompanied by point mutations, and
their presence in different translocation types within the same clone provides
evidence that some of the different translocation types are derived from each
other rather than being formed de novo. These findings provide
insight into eukaryotic genome instability, especially the formation of
translocations and the sources of intraclonal heterogeneity, both of which are
often associated with human cancers
Direct observation of topoisomerase IA gate dynamics
Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6βnm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA
Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1
Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome
Genome-Wide Analysis of Heteroduplex DNA in Mismatch RepairβDeficient Yeast Cells Reveals Novel Properties of Meiotic Recombination Pathways
Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 Γ S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMRβproficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with transβhDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates
Human MLH1 Protein Participates in Genomic Damage Checkpoint Signaling in Response to DNA Interstrand Crosslinks, while MSH2 Functions in DNA Repair
DNA interstrand crosslinks (ICLs) are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA) is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSΞ± and MutSΞ² mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents
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