scholarly journals Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies

2020 ◽  
Vol 6 (7) ◽  
pp. eaay4453
Author(s):  
A. Castañeda-García ◽  
I. Martín-Blecua ◽  
E. Cebrián-Sastre ◽  
A. Chiner-Oms ◽  
M. Torres-Puente ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mycobacterium Smegmatis ◽  
Genome Stability ◽  
Structural Homology ◽  
Spontaneous Mutations ◽  
Wild Type ◽  
Key Factor ◽  
Maintain Genome Stability ◽  
Mmr Proteins

The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution.

scholarly journals Role for Mismatch Repair Proteins Msh2, Mlh1, and Pms2 in Immunoglobulin Class Switching Shown by Sequence Analysis of Recombination Junctions

2002 ◽  
Vol 195 (3) ◽  
pp. 367-373 ◽  
Author(s):  
Carol E. Schrader ◽  
Joycelyn Vardo ◽  
Janet Stavnezer
Keyword(s):  
B Cells ◽  
Mismatch Repair ◽  
Type B ◽  
Wild Type ◽  
Class Switching ◽  
Class Switch ◽  
Mmr Proteins

B cells from mice deficient in mismatch repair (MMR) proteins show decreased ability to undergo class switch recombination in vitro and in vivo. The deficit is not accompanied by any reduction in cell viability or alterations in the cell cycle in B cells cultured in vitro. To assess the role of MMR in switching we examined the nucleotide sequences of Sμ-Sγ3 recombination junctions in splenic B cells induced in culture to switch to IgG3. The data demonstrate clear differences in the sequences of switch junctions in wild-type B cells in comparison with Msh2-, Mlh1-, and Pms2-deficient B cells. Sequences of switch junctions from Msh2-deficient cells showed decreased lengths of microhomology between Sμ and Sγ3 relative to junctions from wild-type cells and an increase in insertions, i.e., nucleotides which do not appear to be derived from either the Sμ or Sγ3 parental sequence. By contrast, 23% of junctions from Mlh1- and Pms2-deficient cells occurred at unusually long stretches of microhomology. The data indicate that MMR proteins are directly involved in class switching and that the role of Msh2 differs from that of Mlh1 and Pms2.

scholarly journals Highly Mismatched Molecules Resembling Recombination Intermediates Efficiently Transform Mismatch Repair Proficient Escherichia coli

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 29-38
Author(s):  
James Westmoreland ◽  
Gregory Porter ◽  
Miroslav Radman ◽  
Michael A Resnick
Keyword(s):  
Mismatch Repair ◽  
Sequence Divergence ◽  
Strand Exchange ◽  
Wild Type ◽  
E Coli ◽  
Key Factor ◽  
Homeologous Recombination ◽  
Related Dnas ◽  
Heteroduplex Formation

The ability of related DNAs to undergo recombination decreases with increased sequence divergence. Mismatch repair has been proposed to be a key factor in preventing homeologous recombination; however, the contribution of mismatch repair is not universal. Although mismatch repair has been proposed to act by preventing strand exchange and/or inactivating multiply mismatched heteroduplexes, there has been no systematic study to determine at what step(s) in recombination mismatch repair acts in vivo. Since heteroduplex is a commonly proposed intermediate in many models of recombination, we have investigated the consequences of mismatch repair on plasmids that are multiply mismatched in heteroduplex structures that are similar to those that might arise during recombination. Plasmids containing multiply mismatched regions were transformed into wild-type and Mut–  Eschericia coli mutants. There was only a 30–40% reduction in transformation of Mut+ as compared to mutS and mutL strains for DNAs containing an 18% mismatched heteroduplex. The products obtained from mutS hosts differed from those obtained from Mut+ hosts in that there were many more colonies containing mixtures of two plasmids, due to survival of both strands of the heteroduplex. There were nearly 10 times more recombinants obtained from the mutS as compared to the wild-type host. Based on these results and those from other studies with E. coli and yeast, we propose that the prevention of recombination between highly diverged DNAs may be at a step earlier than heteroduplex formation.

scholarly journals Mismatch Repair Proteins Regulate Heteroduplex Formation during Mitotic Recombination in Yeast

1998 ◽  
Vol 18 (11) ◽  
pp. 6525-6537 ◽  
Author(s):  
Wenliang Chen ◽  
Sue Jinks-Robertson
Keyword(s):  
Gene Conversion ◽  
Mismatch Repair ◽  
Sister Chromatid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sequence Data ◽  
Mitotic Recombination ◽  
Assay System ◽  
Wild Type ◽  
Mmr Proteins

Mismatch repair (MMR) proteins actively inhibit recombination between diverged sequences in both prokaryotes and eukaryotes. Although the molecular basis of the antirecombination activity exerted by MMR proteins is unclear, it presumably involves the recognition of mismatches present in heteroduplex recombination intermediates. This recognition could be exerted during the initial stage of strand exchange, during the extension of heteroduplex DNA, or during the resolution of recombination intermediates. We previously used an assay system based on 350-bp inverted-repeat substrates to demonstrate that MMR proteins strongly inhibit mitotic recombination between diverged sequences inSaccharomyces cerevisiae. The assay system detects only those events that reverse the orientation of the region between the recombination substrates, which can occur as a result of either intrachromatid crossover or sister chromatid conversion. In the present study we sequenced the products of mitotic recombination between 94%-identical substrates in order to map gene conversion tracts in wild-type versus MMR-defective yeast strains. The sequence data indicate that (i) most recombination occurs via sister chromatid conversion and (ii) gene conversion tracts in an MMR-defective strain are significantly longer than those in an isogenic wild-type strain. The shortening of conversion tracts observed in a wild-type strain relative to an MMR-defective strain suggests that at least part of the antirecombination activity of MMR proteins derives from the blockage of heteroduplex extension in the presence of mismatches.

scholarly journals SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

2016 ◽  
Vol 27 (9) ◽  
pp. 1500-1510 ◽  
Author(s):  
Kentaro Ohkuni ◽  
Yoshimitsu Takahashi ◽  
Alyona Fulp ◽  
Josh Lawrimore ◽  
Wei-Chun Au ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Genome Stability ◽  
Critical Role ◽  
Histone H3 ◽  
E3 Ligases ◽  
Physiological Conditions ◽  
Histone H3 Variant ◽  
Maintain Genome Stability

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.

scholarly journals MiR858b Inhibits Proanthocyanidin Accumulation by the Repression of DkMYB19 and DkMYB20 in Persimmon

2020 ◽  
Vol 11 ◽  
Author(s):  
Sichao Yang ◽  
Meng Zhang ◽  
Liqing Xu ◽  
Zhengrong Luo ◽  
Qinglin Zhang
Keyword(s):  
Expression Patterns ◽  
Transient Transformation ◽  
Structural Genes ◽  
Wild Type ◽  
Rna Ligase ◽  
Key Factor ◽  
R2r3 Myb ◽  
Posttranscriptional Level

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in persimmon.

Maternal Effect for DNA Mismatch Repair in the Mouse

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 271-277
Author(s):  
Vanessa E Gurtu ◽  
Shelly Verma ◽  
Allie H Grossmann ◽  
R Michael Liskay ◽  
William C Skarnes ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Maternal Effect ◽  
Genome Stability ◽  
Germ Line ◽  
Dna Mismatch Repair ◽  
Surprising Result ◽  
Wild Type ◽  
Repair Gene ◽  
Dna Mismatch ◽  
Dna Repair Gene

Abstract DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of ~10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F1 progeny from Pms2−/− female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was ~9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.

scholarly journals Mismatch Repair: From Preserving Genome Stability to Enabling Mutation Studies in Real-Time Single Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1535
Author(s):  
Marina Elez
Keyword(s):  
Real Time ◽  
Mismatch Repair ◽  
Genetic Information ◽  
Genome Stability ◽  
Direct Evidence ◽  
Single Cells ◽  
Living Cells ◽  
Spontaneous Mutations ◽  
Mismatch Recognition

Mismatch Repair (MMR) is an important and conserved keeper of the maintenance of genetic information. Miroslav Radman’s contributions to the field of MMR are multiple and tremendous. One of the most notable was to provide, along with Bob Wagner and Matthew Meselson, the first direct evidence for the existence of the methyl-directed MMR. The purpose of this review is to outline several aspects and biological implications of MMR that his work has helped unveil, including the role of MMR during replication and recombination editing, and the current understanding of its mechanism. The review also summarizes recent discoveries related to the visualization of MMR components and discusses how it has helped shape our understanding of the coupling of mismatch recognition to replication. Finally, the author explains how visualization of MMR components has paved the way to the study of spontaneous mutations in living cells in real time.

scholarly journals ALBA protein complex reads genic R-loops to maintain genome stability in Arabidopsis

2019 ◽  
Vol 5 (5) ◽  
pp. eaav9040 ◽  
Author(s):  
Wei Yuan ◽  
Jincong Zhou ◽  
Jinjin Tong ◽  
Wanqing Zhuo ◽  
Lishuan Wang ◽  
...  
Keyword(s):  
Protein Complex ◽  
Genome Stability ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Single Stranded Dna ◽  
Cellular Processes ◽  
Dna Damaging Agents ◽  
Maintain Genome Stability

The R-loop, composed of a DNA-RNA hybrid and the displaced single-stranded DNA, regulates diverse cellular processes. However, how cellular R-loops are recognized remains poorly understood. Here, we report the discovery of the evolutionally conserved ALBA proteins (AtALBA1 and AtALBA2) functioning as the genic R-loop readers in Arabidopsis. While AtALBA1 binds to the DNA-RNA hybrid, AtALBA2 associates with single-stranded DNA in the R-loops in vitro. In vivo, these two proteins interact and colocalize in the nucleus, where they preferentially bind to genic regions with active epigenetic marks in an R-loop–dependent manner. Depletion of AtALBA1 or AtALBA2 results in hypersensitivity of plants to DNA damaging agents. The formation of DNA breaks in alba mutants originates from unprotected R-loops. Our results reveal that the AtALBA1 and AtALBA2 protein complex is the genic R-loop reader crucial for genome stability in Arabidopsis.

scholarly journals MISMATCH REPAIR MUTATIONS OF ESCHERICHIA COLI K12 ENHANCE TRANSPOSON EXCISION

Genetics ◽  
1985 ◽  
Vol 109 (1) ◽  
pp. 3-19
Author(s):  
Victoria Lundblad ◽  
Nancy Kleckner
Keyword(s):  
Mismatch Repair ◽  
Excision Repair ◽  
General Mechanism ◽  
Mismatch Repair Genes ◽  
Spontaneous Mutations ◽  
E Coli ◽  
Mismatch Correction ◽  
Repair Pathways ◽  
Repair Genes

ABSTRACT Excision of the prokaryotic transposon Tn 10 is a host-mediated process that occurs in the absence of recA function or any transposon-encoded functions. To determine which host functions might play a role in transposon excision, we have isolated 40 mutants of E. coli K12, designated tex, which increase the frequency of Tn 10 precise excision. Three of these mutations (texA) have been shown to qualitatively alter RecBC function. We show that 21 additional tex mutations with a mutator phenotype map to five genes previously identified as components of a methylation-directed pathway for repair of base pair mismatches: uvrD, mutH, mutL, mutS and dam. Previously identified alleles of these genes also have a Tex phenotype.—Several other E. coli mutations affecting related functions have been analyzed for their effects on Tn10 excision. Other mutations affecting the frequency of spontaneous mutations (mutT, polA, ung), different excision repair pathways (uvrA, uvrB) or the state of DNA methylation (dcm) have no effect on Tn 10 excision. Mutations ssb-113 and mutD5, however, do increase Tn 10 excision.—The products of the mismatch correction genes probably function in a coordinated way during DNA repair in vivo. Thus, mutations in these genes might also enhance transposon excision by a single general mechanism. Alternatively, since mutations in each gene have qualitatively and quantitatively different effects on transposon excision, defects in different mismatch repair genes may enhance excision by different mechanisms.

scholarly journals The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Suting Chen ◽  
Tianlu Teng ◽  
Shuan Wen ◽  
Tingting Zhang ◽  
Hairong Huang
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Morphological Changes ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Colony Morphology ◽  
Wall Structure ◽  
Wild Type

Abstract Background: The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant ( aceE -mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis , basonym Mycobacterium smegmatis ( M. smegmatis ). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis. Results: We observed that the colony morphology of aceE -mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis . Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE -mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE -mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE -mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE -mut strain whereas complementation of the aceE -mut with a wild-type aceE gene restored the composition of MA. Conclusions: Over all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.

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