Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: a simple genetic system to assay MLH1 function.
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ABSTRACT:
SUBMITTER: Quaresima B
PROVIDER: S-EPMC1223262 | biostudies-other | 2003 Apr
REPOSITORIES: biostudies-other
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Project description:The ternary complex comprising MutS, MutL, and DNA is a key intermediate in DNA mismatch repair. We used chemical cross-linking and fluorescence resonance energy transfer (FRET) to study the interaction between MutS and MutL and to shed light onto the structure of this complex. Via chemical cross-linking, we could stabilize this dynamic complex and identify the structural features of key events in DNA mismatch repair. We could show that in the complex between MutS and MutL the mismatch-binding and connector domains of MutS are in proximity to the N-terminal ATPase domain of MutL. The DNA- and nucleotide-dependent complex formation could be monitored by FRET using single cysteine variants labeled in the connector domain of MutS and the transducer domain of MutL, respectively. In addition, we could trap MutS after an ATP-induced conformational change by an intramolecular cross-link between Cys-93 of the mismatch-binding domain and Cys-239 of the connector domain.
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Project description:BACKGROUND:Acinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to investigate DNA mismatch repair, a mechanism for repairing mismatched bases. We used the mutS deletion mutant (XH439) and mutL deletion mutant (XH440), and constructed a mutS mutL double deletion mutant (XH441) to investigate the role of the mismatch repair system in A. baylyi. RESULTS:We determined the survival rates after UV irradiation and measured the mutation frequencies, rates and spectra of wild-type ADP1 and mutSL mutant via rifampin resistance assay (RifR assay) and experimental evolution. In addition, transformation efficiencies of genomic DNA in ADP1 and its three mutants were determined. Lastly, the relative growth rates of the wild type strain, three constructed deletion mutants, as well as the rifampin resistant mutants obtained from RifR assays, were measured. All three mutants had higher survival rates after UV irradiation than wild type, especially the double deletion mutant. Three mutants showed higher mutation frequencies than ADP1 and favored transition mutations in RifR assay. All three mutants showed increased mutation rates in the experimental evolution. However, only XH439 and XH441 had higher mutation rates than the wild type strain in RifR assay. XH441 showed higher transformation efficiency than XH438 when donor DNA harbored transition mutations. All three mutants showed higher growth rates than wild-type, and these four strains displayed higher growth rates than almost all their rpoB mutants. The growth rate results showed different amino acid mutations in rpoB resulted in different extents of reduction in the fitness of rifampin resistant mutants. However, the fitness cost brought by the same mutation did not vary with strain background. CONCLUSIONS:We demonstrated that inactivation of both mutS and mutL increased the mutation rates and frequencies in A. baylyi, which would contribute to the evolution and acquirement of rifampicin resistance. The mutS deletion is also implicated in increased mutation rates and frequencies, suggesting that MutL may be activated even in the absence of mutS. The correlation between fitness cost and rifampin resistance mutations in A. baylyi is firstly established.
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Project description:DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL's endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS-MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.
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Project description:DNA mismatch repair (MMR) relies on MutS and MutL ATPases for mismatch recognition and strand-specific nuclease recruitment to remove mispaired bases in daughter strands. However, whether the MutS-MutL complex coordinates MMR by ATP-dependent sliding on DNA or protein-protein interactions between the mismatch and strand discrimination signal is ambiguous. Using functional MMR assays and systems preventing proteins from sliding, we show that sliding of human MutSα is required not for MMR initiation, but for final mismatch removal. MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5' to the mismatch. Exonuclease 1 (Exo1) is then recruited to the nick and conducts 5' → 3' excision. ATP-dependent MutSα dissociation from the mismatch is necessary for Exo1 to remove the mispaired base when the excision reaches the mismatch. Therefore, our study has resolved a long-standing puzzle, and provided new insights into the mechanism of MMR initiation and mispair removal.
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Project description:Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis. Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10-40% of related sporadic tumours. However, the collaborative mechanics of MSH and MLH/PMS proteins have not been resolved in any organism. We visualized Escherichia coli (Ec) ensemble mismatch repair and confirmed that EcMutS mismatch recognition results in the formation of stable ATP-bound sliding clamps that randomly diffuse along the DNA with intermittent backbone contact. The EcMutS sliding clamps act as a platform to recruit EcMutL onto the mismatched DNA, forming an EcMutS-EcMutL search complex that then closely follows the DNA backbone. ATP binding by EcMutL establishes a second long-lived DNA clamp that oscillates between the principal EcMutS-EcMutL search complex and unrestricted EcMutS and EcMutL sliding clamps. The EcMutH endonuclease that targets mismatch repair excision only binds clamped EcMutL, increasing its DNA association kinetics by more than 1,000-fold. The assembly of an EcMutS-EcMutL-EcMutH search complex illustrates how sequential stable sliding clamps can modulate one-dimensional diffusion mechanics along the DNA to direct mismatch repair.
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Project description:The identification and analysis of the Pseudomonas avellanae mismatch repair system (MMR) were performed via sequencing and cloning the mutS and mutL genes and then analyzing the characteristics of the corresponding proteins studying their function and biological role in an E. coli heterologous system. In these studies, the P. avellanae MutS and MutL proteins were shown to localise at the nucleoid level, in a MutS-dependent manner as far as MutL is concerned, and were also able to complement the defect observed in both the mutS and mutL knockout strains of E. coli. In addition, their ability to form both homo and heterodimers between each other was shown by using the prokaryotic two-hybrid assay. Our results represent a first step to elucidate the MMR mechanism in plant pathogenic pseudomonads since the MMR genes were identified in P. syringae pathovars but there was no evidence on their action as effective repair products.
| S-EPMC3367300 | biostudies-literature
Project description:A shared paradigm of mismatch repair (MMR) across biology depicts extensive exonuclease-driven strand-specific excision that begins at a distant single-stranded DNA (ssDNA) break and proceeds back past the mismatched nucleotides. Historical reconstitution studies concluded that Escherichia coli (Ec) MMR employed EcMutS, EcMutL, EcMutH, EcUvrD, EcSSB and one of four ssDNA exonucleases to accomplish excision. Recent single-molecule images demonstrated that EcMutS and EcMutL formed cascading sliding clamps on a mismatched DNA that together assisted EcMutH in introducing ssDNA breaks at distant newly replicated GATC sites. Here we visualize the complete strand-specific excision process and find that long-lived EcMutL sliding clamps capture EcUvrD helicase near the ssDNA break, significantly increasing its unwinding processivity. EcSSB modulates the EcMutL-EcUvrD unwinding dynamics, which is rarely accompanied by extensive ssDNA exonuclease digestion. Together these observations are consistent with an exonuclease-independent MMR strand excision mechanism that relies on EcMutL-EcUvrD helicase-driven displacement of ssDNA segments between adjacent EcMutH-GATC incisions.
| S-EPMC6876574 | biostudies-literature
Project description:Mutations in mutS and mutL, which encode DNA mismatch repair (MMR) proteins, can confer hypermutator phenotypes and may facilitate the emergence of mutational antibiotic resistance in bacteria. Linezolid-resistant enterococci (LRE) rarely emerge during therapy and contain mutations in 23S rRNA genes. As enterococci with defective MMR could be prone to the development of oxazolidinone resistance mutations, we investigated 13 clinical isolates of Enterococcus faecium, including 2 LRE, for mutations in mutSL. A 4,944-bp fragment spanning mutSL was sequenced from two pairs of linezolid-resistant (MICs, 64 micro g/ml) and linezolid-susceptible (MICs, 2 micro g/ml) E. faecium isolates (one pair from Austria and one pair from the United Kingdom) identical by pulsed-field gel electrophoresis. The pairs represented distinct strains in which linezolid resistance had emerged during therapy. The MutSL peptides of all four isolates had amino acid substitutions compared with the sequence of E. faecium strain DO (used for genome sequencing). These were Val352Ile (one pair of isolates only) and Met628Leu in MutS and Leu387Pro, Tyr406Phe, Thr415Ser, Phe427Leu, and Phe565Ile in MutL. The significance of these changes remains unknown; these isolates did not show a demonstrable hypermutator phenotype. The same substitutions were found in two of nine geographically diverse linezolid-susceptible enterococcal isolates; the other seven isolates had MutSL sequences identical to that of strain DO. Multilocus sequence typing revealed that all isolates with alternate MutSL peptides belonged to a distinct lineage of a prevalent E. faecium clonal complex, designated CC17. Further studies are needed to investigate the prevalence of these MutSL mutations and their possible roles in the emergence of E. faecium strains resistant to oxazolidinones and other antibiotic classes.
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Project description:Escherichia coli MutS forms a mispair-dependent ternary complex with MutL that is essential for initiating mismatch repair (MMR) but is structurally uncharacterized, in part owing to its dynamic nature. Here, we used hydrogen/deuterium exchange mass spectrometry and other methods to identify a region in the connector domain (domain II) of MutS that binds MutL and is required for mispair-dependent ternary complex formation and MMR. A structurally conserved region in Msh2, the eukaryotic homolog, was required for formation of a mispair-dependent Msh2-Msh6-Mlh1-Pms1 ternary complex. These data indicate that the connector domain of MutS and Msh2 contains the interface for binding MutL and Mlh1-Pms1, respectively, and support a mechanism whereby mispair and ATP binding induces a conformational change that allows the MutS and Msh2 interfaces to interact with their partners.
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Project description:The mismatch repair (MMR) complex is composed of MutS? (MSH2-MSH6) and MutL? (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. O 6-Methylguanine is produced by treatment with alkylating agents, such as N-methyl-N-nitrosourea (MNU), and during DNA replication forms a DNA mismatch (i.e. an O 6-methylguanine/thymine pair) and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear. In this study, we provide evidence that the chromatin-regulatory and ATP-dependent nucleosome-remodeling protein SMARCAD1 is involved in the induction of MMR-dependent apoptosis in human cells. Unlike control cells, SMARCAD1-knockout cells (?SMARCAD1) were MNU-resistant, and the appearance of a sub-G1 population and caspase-9 activation were significantly suppressed in the ?SMARCAD1 cells. Furthermore, the MNU-induced mutation frequencies were increased in these cells. Immunoprecipitation analyses revealed that the recruitment of MutL? to chromatin-bound MutS?, observed in SMARCAD1-proficient cells, is suppressed in ?SMARCAD1 cells. Of note, the effect of SMARCAD1 on the recruitment of MutL? exclusively depended on the ATPase activity of the protein. On the basis of these findings, we propose that SMARCAD1 induces apoptosis via its chromatin-remodeling activity, which helps recruit MutL? to MutS? on damaged chromatin.
| S-EPMC6983851 | biostudies-literature