The Escherichia coli MutL protein stimulates binding of Vsr and MutS to heteroduplex DNA

Drotschmann, Karin; Aronshtam, Alexander; Fritz, Hans-Joachim; Marinus, Martin

doi:10.1093/nar/26.4.948

Cited by 95 publications

(65 citation statements)

References 37 publications

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“…Examination of the interaction of MutS and MutL with a mismatch DNA in the presence of ATP reveals that MutL blocks the migration of MutS and results in a supershift on polyacryl- amide gels consistent with a ternary complex composed of MutS, MutL, and heteroduplex DNA. Such a ternary complex of E. coli MMR proteins is consistent with experiments employing co-immunoprecipitation (8), surface plasmon resonance (10), affinity chromatography (25), gel electrophoresis (30), and DNase I footprinting (7).…”

Section: Discussionsupporting

confidence: 79%

Interaction of Escherichia coli MutS and MutL at a DNA Mismatch

Schofield

Nayak

Scott

et al. 2001

Journal of Biological Chemistry

129

159

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MutS and MutL are both required to activate downstream events in DNA mismatch repair. We examined the rate of dissociation of MutS from a mismatch using linear heteroduplex DNAs or heteroduplexes blocked at one or both ends by four-way DNA junctions in the presence and absence of MutL. In the presence of ATP, dissociation of MutS from linear heteroduplexes or heteroduplexes blocked at only one end occurs within 15 s. When both duplex ends are blocked, MutS remains associated with the DNA in complexes with half-lives of 30 min. DNase I footprinting of MutS complexes is consistent with migration of MutS throughout the DNA duplex region. When MutL is present, it associates with MutS and prevents ATP-dependent migration away from the mismatch in a manner that is dependent on the length of the heteroduplex. The rate and extent of mismatch-provoked cleavage at hemimethylated GATC sites by MutH in the presence of MutS, MutL, and ATP are the same whether the mismatch and GATC sites are in cis or in trans. These results suggest that a MutS-MutL complex in the vicinity of a mismatch is involved in activating MutH.Misincorporation of bases during DNA replication that escapes proofreading results in the formation of mismatches, either unpaired or mispaired bases. The DNA mismatch repair pathway targets these mismatches for correction and contributes as much as 1000-fold to the overall fidelity of DNA replication (reviewed in Refs. 1 and 2). The importance of this repair pathway is highlighted by the finding that mutations in mismatch repair genes segregate with the cancer predisposition syndromes hereditary nonpolyposis colon cancer and familial colorectal cancer (reviewed in Refs. 3 and 4). In addition, inactivation of mismatch repair genes by promoter methylation has been documented in some sporadic tumors (reviewed in Ref. 1).Much of our current understanding of mismatch repair stems from studies of the Escherichia coli methyl-directed mismatch repair pathway where repair has been reconstituted in vitro using purified proteins (1, 2). MutS recognizes and binds to seven of eight possible base pair mismatches as well as one to four unpaired bases. These mismatches arise by misincorporation of deoxynucleotides or template slippage, respectively. In addition to binding DNA, all MutS proteins have an intrinsic ATPase activity and are members of the ATP binding cassette transporter superfamily (5). Recognition of the mismatch by MutS triggers subsequent steps of mismatch repair in which MutS together with MutL protein and ATP activates an endonuclease, MutH, that cleaves a transiently unmethylated daughter strand at hemimethylated GATC sites, thereby conferring strand specificity on the repair process. The mismatchprovoked strand scission constitutes a point of entry for helicase II (UvrD) and exonucleases that excise the daughter strand in the region spanning the GATC site and the mis- A critical problem in mismatch repair is understanding how binding to a mismatch by MutS triggers downstream repair events such as the activat...

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Section: Discussionsupporting

confidence: 79%

Interaction of Escherichia coli MutS and MutL at a DNA Mismatch

Schofield

Nayak

Scott

et al. 2001

Journal of Biological Chemistry

129

159

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“…It was indeed observed that the inhibition of in vitro strand exchange between 3% diverged DNA substrates increases with rising concentration of MutL protein at a constant level of MutS protein (L. Worth, Jr., personal communication). Similarly, the probability at which MutS binds to heteroduplex DNA containing a single mismatch in vitro increases with rises in MutL (36).…”

Section: Discussionmentioning

confidence: 98%

The frequency and structure of recombinant products is determined by the cellular level of MutL

Elez

Radman

Matić

2007

Proc. Natl. Acad. Sci. U.S.A.

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The presence of repeated DNA sequences is a genomic liability, because interrepeat recombination can result in chromosomal rearrangements. The mismatch repair system prevents recombination between nonidentical repeats, but the mechanism of antirecombination has not been established. Although the MutS protein binds to base pair mismatches in heteroduplex DNA, the role of the MutL protein in preventing recombination is unknown. In a screen designed to identify new cellular functions that suppress deletion formation involving nonidentical DNA repeats, we isolated a mutL mutant having a separation-of-function phenotype. The mutant showed an increased frequency of deletions but not of mutations. The split phenotype is due to a decreased MutL level, indicating that recombination, but not replication editing, is highly sensitive to MutL level. By altering the MutL level, we found that the frequency of deletion-generating recombination is inversely related to the amount of cellular MutL. DNA sequence analysis of the recombined repeats shows that the tolerance of base pair mismatches in heteroduplex DNA is also inversely correlated with MutL level. Unlike recombination, correction of misincorporation errors by mismatch repair is insensitive to fluctuations in MutL level. Overproduction of MutS does not affect either of these phenotypes, suggesting that, unlike MutL, MutS is not limiting for mismatch repair activities. These results indicate that MutL (i) determines effective DNA homology in recombination processes and (ii) fine tunes the process of deletion formation involving repeated, diverged DNA sequences.deletions ͉ DNA repeats ͉ mismatch repair ͉ recombination ͉ replication P rokaryotic genomes are riddled with DNA sequence repeats, which are found in intergenic regions, genes, and transposable elements (1). Repeats are even more abundant in eukaryotic genomes (2), and at least 34% of the human genome consists of such sequences (3). The presence of dispersed repeated DNA sequences is a genetic liability because interrepeat recombination can cause deletions, duplications, inversions or translocations, depending on the configuration and orientation of the repeat units. In humans, recombination between repeats is at the origin of disease-causing deletions, such as ␣-thalassemias, Duchenne muscular dystrophy, and familial hypercholesterolemia (4-7).Recombination between repeats is constantly initiated by DNA damage and/or DNA replication blockage that results from exogenous and endogenous genomic insults. Therefore, natural selection for genome stability has resulted in the emergence of mechanisms that prevent interrepeat recombination. Newly arising repeats are identical at the DNA sequence level, but established repeats have usually diverged (8). The degree and distribution of sequence divergence are structural parameters that influence recombination between repeats because they affect the activity of recombination enzymes and determine whether the antirecombinogenic activity of mismatch repair (MMR) proteins is trig...

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“…Protein Purification-Hexameric histidine-tagged MutS protein was prepared as previously described (39). The host strain was BL21 ( DE3) (Novagen) and the expression vector pMQ382 was kindly provided by Dr. M. G. Marinus.…”

Section: Methodsmentioning

confidence: 99%

Binding Discrimination of MutS to a Set of Lesions and Compound Lesions (Base Damage and Mismatch) Reveals Its Potential Role as a Cisplatin-damaged DNA Sensing Protein

Fourrier

Brooks²,

Malinge³

2003

Journal of Biological Chemistry

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The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin. It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions. This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis. Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions. The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate. The rate of association, k on, for binding to the 1,2-d(GpG) adduct was 3.1 ؋ 10 4 M ؊1 s ؊1 and the specificity of binding was essentially dependent on k off . DNA duplexes containing a single 1,2-d(ApG), 1,3-d(GpCpG) adduct, and an interstrand cross-link of cisplatin were not preferentially recognized. Among 12 DNA substrates, each containing a different cisplatin compound lesion derived from replicative misincorporation of one base opposite either of the 1,2-intrastrand adducts, 10 were specifically recognized including those that are more likely formed in vivo based on cisplatin mutation spectra. Moreover, among these lesions, two compound lesions formed when an adenine was misincorporated opposite a 1,2-d(GpG) adduct were not substrates for the MutYdependent mismatch repair pathway. The ability of MutS to sense differentially various platinated DNA substrates suggests that cisplatin compound lesions formed during misincorporation of a base opposite either adducted base of both 1,2-intrastrand cross-links are more plausible critical lesions for MMR-mediated cisplatin cytotoxicity.cis-Diamminedichloroplatinum(II) (cisplatin) 1 is among the most widely used anticancer chemotherapeutic agents in the treatment of many human tumors, particularly testicular and ovarian tumors (1). In the reaction between DNA and cisplatin, different types of bifunctional adducts are formed that are thought to be the key toxic lesions (2-4). Although these adducts are responsible for a variety of cellular responses including replication and/or transcription inhibition, there is not yet a clear understanding of the molecular mechanisms linking the formation of adducts and cisplatin-induced apoptosis (5, 6). Cisplatin reacts preferentially with the N-7 atoms of purine residues in DNA. The major adducts (90%) are 1,2-intrastrand cross-links at d(GpG) and d(ApG) sites and the minor adducts correspond to 1,3-intrastrand cross-links at d(GpNpG) (N being a nucleotide residue) and interstrand cross-links formed at d(GpC/GpC) sites (7-10). Once formed, cisplatin lesions such as the major 1,2-intrastrand cross-links can undergo replication bypass in cells treated with cisplatin (11,12). Be...

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The Escherichia coli MutL protein stimulates binding of Vsr and MutS to heteroduplex DNA

Cited by 95 publications

References 37 publications

Interaction of Escherichia coli MutS and MutL at a DNA Mismatch

Interaction of Escherichia coli MutS and MutL at a DNA Mismatch

The frequency and structure of recombinant products is determined by the cellular level of MutL

Binding Discrimination of MutS to a Set of Lesions and Compound Lesions (Base Damage and Mismatch) Reveals Its Potential Role as a Cisplatin-damaged DNA Sensing Protein

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