scholarly journals Mapping the Polarity of Changes That Occur in Interrupted CAG Repeat Tracts in Yeast

1998 ◽  
Vol 18 (8) ◽  
pp. 4597-4604 ◽  
Author(s):  
Debra J. Maurer ◽  
Brennon L. O’Callaghan ◽  
Dennis M. Livingston
Keyword(s):  
Mismatch Repair ◽  
Trinucleotide Repeat ◽  
Cag Repeat ◽  
Yeast Cells ◽  
Wild Type ◽  
Repeat Tract ◽  
Repeat Units ◽  
Length Changes ◽  
Mutant Cells ◽  
Lagging Strand

ABSTRACT To explore the mechanisms by which CAG trinucleotide repeat tracts undergo length changes in yeast cells, we examined the polarity of alterations with respect to an interrupting CAT trinucleotide near the center of the tract. In wild-type cells, in which most tract changes are large contractions, the changes that retain the interruption are biased toward the 3′ end of the repeat tract (in reference to the direction of lagging-strand synthesis). In rth1/rad27mutant cells that are defective in Okazaki fragment maturation, the tract expansions are biased to the 5′ end of the repeat tract, while the tract contractions that do not remove the interruption occur randomly on either side of the interruption. In msh2 mutant cells that are defective in the mismatch repair machinery, neither the small changes of one or two repeat units nor the larger contractions attributable to this mutation are biased to either side of the interruption. The results of this study are discussed in terms of the molecular paths leading to expansions and contractions of repeat tracts.

scholarly journals Stabilizing Effects of Interruptions on Trinucleotide Repeat Expansions in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Michael L. Rolfsmeier ◽  
Robert S. Lahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Trinucleotide Repeat ◽  
Primary Factor ◽  
Yeast Cells ◽  
Repeat Expansions ◽  
Yeast Genetic ◽  
Hairpin Formation ◽  
Lagging Strand ◽  
Genetic Assay

ABSTRACT In most trinucleotide repeat (TNR) diseases, the primary factor determining the likelihood of expansions is the length of the TNR. In some diseases, however, stable alleles contain one to three base pair substitutions that interrupt the TNR tract. The unexpected stability of these alleles compared to the frequent expansions of perfect TNRs suggested that interruptions somehow block expansions and that expansions occur only upon loss of at least one interruption. The work in this study uses a yeast genetic assay to examine the mechanism of stabilization conferred by two interruptions of a 25-repeat tract. Expansion rates are reduced up to 90-fold compared to an uninterrupted allele. Stabilization is greatest when the interruption is replicated early on the lagging strand, relative to the rest of the TNR. Although expansions are infrequent, they are often polar, gaining new DNA within the largest available stretch of perfect repeats. Surprisingly, interruptions are always retained and sometimes even duplicated, suggesting that expansion in yeast cells can proceed without loss of the interruption. These findings support a stabilization model in which interruptions contribute in cis to reduce hairpin formation during TNR replication and thus inhibit expansion rates.

scholarly journals Human mismatch repair system corrects errors produced during lagging strand replication more effectively

2016 ◽  
Author(s):  
Maria Andrianova ◽  
Georgii A Bazykin ◽  
Sergey Nikolaev ◽  
Vladimir Seplyarskiy
Keyword(s):  
Mismatch Repair ◽  
Mutation Rates ◽  
Repair System ◽  
Wild Type ◽  
Strand Asymmetry ◽  
Exonuclease Domain ◽  
Mismatch Repair System ◽  
Dna Strands ◽  
Leading Strand ◽  
Lagging Strand

Mismatch repair (MMR) is one of the main systems maintaining fidelity of replication. Different effectiveness in correction of errors produced during replication of the leading and the lagging DNA strands was reported in yeast, but this effect is poorly studied in humans. Here, we use MMR-deficient (MSI) and MMR-proficient (MSS) cancer samples to investigate properties of the human MMR. MSI, but not MSS, cancers demonstrate unequal mutation rates between the leading and the lagging strands. The direction of strand asymmetry in MSI cancers matches that observed in cancers with mutated exonuclease domain of polymerase δ, indicating that polymerase δ contributes more mutations than its leading-strand counterpart, polymerase ε. As polymerase δ primarily synthesizes DNA during the lagging strand replication, this implies that mutations produced in wild type cells during lagging strand replication are repaired by the MMR ~3 times more effectively, compared to those produced on the leading strand.

scholarly journals The number of cytokinesis nodes in mitotic fission yeast scales with cell volume

2021 ◽  
Author(s):  
Wasim A Sayyad ◽  
Thomas D Pollard
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Large Population ◽  
Yeast Cells ◽  
Wild Type ◽  
Contractile Ring ◽  
Node Number ◽  
Nmt1 Promoter ◽  
Wide Range ◽  
Mutant Cells

Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here we used the ~140 nm resolution of Airyscan confocal microscopy to resolve a large population of dim, unitary cytokinesis nodes in 3D reconstructions of whole fission yeast cells. Wild-type fission yeast cells make about 200 unitary cytokinesis nodes. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although wide rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The surface density of Pom1 kinase on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, varying protein concentrations with the nmt1 promoter showed that the numbers of nodes increase above a baseline of about 200 with the total cellular concentration of either Pom1 or the kinase Cdr2.

scholarly journals Ribonucleotide incorporation characteristics around yeast autonomously replicating sequences reveal the labor division of replicative DNA polymerases

2020 ◽  
Author(s):  
Penghao Xu ◽  
Francesca Storici
Keyword(s):  
Dna Polymerase ◽  
Genome Instability ◽  
Dna Polymerases ◽  
Yeast Cells ◽  
Yeast Genome ◽  
Wild Type ◽  
Specific Sequence ◽  
Leading Strand ◽  
Mapping Techniques ◽  
Lagging Strand

ABSTRACTRibonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARS’s) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARS’s, highlighting the Pol δ - Pol ε handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol δ and Pol ε.

scholarly journals Androgen Receptor Gene CAG Trinucleotide Repeats in Anovulatory Infertility and Polycystic Ovaries

2000 ◽  
Vol 85 (9) ◽  
pp. 3484-3488 ◽  
Author(s):  
Amparo Mifsud ◽  
Sylvia Ramirez ◽  
E. L. Yong
Keyword(s):  
Androgen Receptor ◽  
Trinucleotide Repeat ◽  
Age Of Onset ◽  
Receptor Gene ◽  
Normal Serum ◽  
Cag Repeat ◽  
Polycystic Ovaries ◽  
Repeat Tract ◽  
Serum Androgens ◽  
Anovulatory Infertility

Abstract Hyperandrogenism is currently thought to be central to the pathogenesis of polycystic ovarian syndrome (PCOS), a common endocrine disorder in premenopausal women characterized by irregular menstruation and anovulatory infertility. Although hyperandrogenism is characteristic, some women with PCOS have normal serum androgen levels. All androgens act through the X-linked androgen receptor (AR), the N-terminal domain of which contains a polyglutamine tract encoded by a highly polymorphic CAG trinucleotide repeat tract. Recently, variations in this CAG microsatellite tract, while remaining within the normal polymorphic range (11–38 CAGs), have been inversely correlated with receptor activity. Thus, short tracts are associated with high intrinsic AR activity and increased severity and earlier age of onset of the androgen-regulated tumor prostate cancer, whereas longer CAG tracts are associated with low AR activity and oligospermic infertility. To investigate the role of the CAG repeat tract in PCOS, we measured its length in 91 patients with ultrasound diagnosis of polycystic ovaries, irregular menstrual cycles, and anovulatory infertility and compared them to 112 control subjects of proven fertility with regular menses. Fluorescent-labeled DNA fragments containing the CAG repeat tract were amplified from leucocytic DNA, and their lengths were compared with internal size markers on an automated DNA Sequencer. There were no differences in the mean CAG length between patients and controls when both alleles were considered together or separately. Because there is a subset of PCOS patients whose serum androgens are normal, we compared differences in CAG length between patients whose serum testosterone (T) levels were below the normal laboratory mean, to those that were higher. There was a trend for a lower mean CAG biallelic length among anovulatory patients with T less than 1.73 nmol/L compared with those whose T was more than 1.73 nmol/L (22.47 ± 0.36 vs. 23.25 ± 0.29). This difference in CAG length between patients with low and high T levels (20.38 ± 0.51 vs. 21.98 ± 0.29) was highly significant (P = 0.004) when only the shorter allele of each individual was considered. Ethnic differences were also evident in our data; Indian subjects had a significantly shorter AR-CAG length compared with Chinese, being 22.08 ± 0.50 and 23.16 ± 0.17, respectively. Our data indicate an association between short CAG repeat length and the subset of anovulatory patients with low serum androgens, suggesting that the pathogenic mechanism of polycystic ovaries in these patients could be due to the increased intrinsic androgenic activity associated with short AR alleles.

scholarly journals Functional Overlap in Mismatch Repair by Human MSH3 and MSH6

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1637-1646 ◽  
Author(s):  
Asad Umar ◽  
John I Risinger ◽  
Warren E Glaab ◽  
Kenneth R Tindall ◽  
J Carl Barrett ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Microsatellite Loci ◽  
Tumor Cell Line ◽  
Mutation Rates ◽  
Wild Type ◽  
Chromosome 5 ◽  
Replication Errors ◽  
Repeat Units ◽  
Human Genes ◽  
Hprt Locus

Abstract Three human genes, hMSH2, hMSH3, and hMSH6, are homologues of the bacterial MutS gene whose products bind DNA mismatches to initiate strand-specific repair of DNA replication errors. Several studies suggest that a complex of hMSH2·hMSH6 (hMutSα) functions primarily in repair of base·base mismatches or single extra bases, whereas a hMSH2·hMSH3 complex (hMutSβ) functions chiefly in repair of heteroduplexes containing two to four extra bases. In the present study, we compare results with a tumor cell line (HHUA) that is mutant in both hMSH3 and hMSH6 to results with derivative clones containing either wild-type hMSH3 or wild-type hMSH6, introduced by microcell-mediated transfer of chromosome 5 or 2, respectively. HHUA cells exhibit marked instability at 12 different microsatellite loci composed of repeat units of 1 to 4 base pairs. Compared to normal cells, HHUA cells have mutation rates at the HPRT locus that are elevated 500-fold for base substitutions and 2400-fold for single-base frameshifts. Extracts of HHUA cells are defective in strand-specific repair of substrates containing base·base mismatches or 1–4 extra bases. Transfer of either chromosome 5 (hMSH3) or 2 (hMSH6) into HHUA cells partially corrects instability at the microsatellite loci and also the substitution and frameshift mutator phenotypes at the HPRT locus. Extracts of these lines can repair some, but not all, heteroduplexes. The combined mutation rate and mismatch repair specificity data suggest that both hMSH3 and hMSH6 can independently participate in repair of replication errors containing base·base mismatches or 1–4 extra bases. Thus, these two gene products share redundant roles in controlling mutation rates in human cells.

scholarly journals Importance of catalase in the adaptive response to hydrogen peroxide: analysis of acatalasaemic Saccharomyces cerevisiae

1996 ◽  
Vol 320 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Shingo IZAWA ◽  
Yoshiharu INOUE ◽  
Akira KIMURA
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Adaptive Response ◽  
Yeast Cells ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Single Mutant ◽  
Mutant Cells ◽  
Similar Growth

Controversy about the importance of catalase in the detoxification of H2O2 in human erythrocytes continues. It has been suggested that catalase has no role in the clearance of H2O2 in erythrocytes. In the present study we investigated the role of catalase in the defence mechanism against oxidative stress using Saccharomyces cerevisiae. S. cerevisiae has two catalases, catalase A and catalase T. We constructed a double mutant (acatalasaemic mutant) unable to produce either catalase A or catalase T, and compared it with wild-type and single-mutant cells. The acatalasaemic mutant cells showed a similar growth rate to wild-type cells under non-oxidative stress conditions, and showed a similar susceptibility to H2O2 stress in the exponential growth phase. The acatalasaemic mutant cells at stationary phase were, however, much more sensitive to H2O2 stress than wild-type and single-mutant cells. Moreover, the ability of acatalasaemic and single-mutant cells to show adaptation to 2 mM H2O2 was distinctly inferior to that of wild-type cells. These results suggest that catalase is not essential for yeast cells under normal conditions, but plays an important role in the acquisition of tolerance to oxidative stress in the adaptive response of these cells.

scholarly journals Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane.

1994 ◽  
Vol 126 (6) ◽  
pp. 1361-1373 ◽  
Author(s):  
L F Sogo ◽  
M P Yaffe
Keyword(s):  
Outer Membrane ◽  
Yeast Cells ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Wild Type ◽  
Giant Mitochondria ◽  
Mitochondrial Distribution ◽  
Rapid Restoration ◽  
Mutant Phenotypes ◽  
Mutant Cells

Yeast cells with the mdm10 mutation possess giant spherical mitochondria and are defective for mitochondrial inheritance. The giant mitochondria display classical features of mitochondrial ultrastructure, yet they appear incapable of movement or division. Genetic analysis indicated that the mutant phenotypes resulted from a single nuclear mutation, and the isolated MDM10 gene restored wild-type mitochondrial distribution and morphology when introduced into mutant cells. MDM10 encodes a protein of 56.2 kD located in the mitochondrial outer membrane. Depletion of Mdm10p from cells led to a condensation of normally extended, tubular mitochondria into giant spheres, and reexpression of the protein resulted in a rapid restoration of normal mitochondrial morphology. These results demonstrate that Mdm10p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria.

scholarly journals Mechanisms of Contact-Mediated Killing of Yeast Cells on Dry Metallic Copper Surfaces

2010 ◽  
Vol 77 (2) ◽  
pp. 416-426 ◽  
Author(s):  
Davide Quaranta ◽  
Travis Krans ◽  
Christophe Espírito Santo ◽  
Christian G. Elowsky ◽  
Dylan W. Domaille ◽  
...  
Keyword(s):  
Copper Ions ◽  
Metallic Copper ◽  
Membrane Damage ◽  
Ion Homeostasis ◽  
Genetic Material ◽  
Copper Ion ◽  
Yeast Cells ◽  
Wild Type ◽  
Copper Surfaces ◽  
Mutant Cells

ABSTRACTSurfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivateCandida albicansandSaccharomyces cerevisiaewithin minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactiveS. cerevisiaeCtr1p (ScCtr1p) copper uptake transporter inSaccharomycesresulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of theC. albicansCrp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing ofCandidamutant cells than of wild-type cells.CandidaandSaccharomycestook up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay withSaccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC2(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated.

scholarly journals Repair of Double-Strand Breaks by Homologous Recombination in Mismatch Repair-Defective Mammalian Cells

2001 ◽  
Vol 21 (8) ◽  
pp. 2671-2682 ◽  
Author(s):  
Beth Elliott ◽  
Maria Jasin
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Strand Breaks ◽  
Mutant Cells

ABSTRACT Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93–101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2 −/− cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.

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