DNA Mismatch Binding Activities Research Articles

In vitro affinity of Deinococcus radiodurans MutS towards mismatched DNA exceeds that of its orthologues from Escherichia coli and Thermus thermophilus

The mismatch binding protein MutS is responsible for the recognition of mispaired and unpaired bases, which is the initial step in DNA repair. Among the MutS proteins most extensively studied in vitro are those derived from Thermus thermophilus, Thermus aquaticus and Escherichia coli. Here, we present the first report on the in vitro examination of DNA mismatch binding activity of MutS protein from Deinococcus radiodurans and confront this with the properties of those from E. coli and T. thermophilus. The analyses which included mobility gel-shift assay, colorimetric and qPCR estimation of MutS-bound DNA clearly showed that D. radiodurans MutS exhibited much higher affinity towards mismatched DNA in vitro than its counterparts from E. coli and T. thermophilus. In addition, D. radiodurans MutS displayed a significantly higher specificity of DNA mismatch binding than the two other orthologues. The specificity expressed as the ratio of mismatched to fully complementary DNA bound reached over 4 and 20-fold higher values for D. radiodurans than for T. thermophilus and E. coli MutS, respectively. The results demonstrate mainly the biotechnological potential of D. radiodurans MutS but the in vitro characteristics of the MutS orthologues could reflect substantial differences in DNA mismatch binding activities existing in vivo.

Differential mechanisms of cadmium and mercury(II)-induced down-regulation of DNA mismatch binding activities in zebrafish (Danio rerio) embryos

Differential mechanisms of cadmium and mercury(II)-induced down-regulation of DNA mismatch binding activities in zebrafish (Danio rerio) embryos

A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5–10 pmol) and DNA (0.1–10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA–protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2–Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S 1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S 2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S 1—but which of these subunits, S 1 or S 2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2–Msh6 heterodimer (in Msh2–Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S 1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S 2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a “ cis” linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2–Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.

DNA mismatch binding activities in Chlorella pyrenoidosa extracts and affinity isolation of G-T mismatch binding proteins

DNA mismatch recognition proteins contained in the extracts of unicellular alga Chlorella pyrenoidosa were isolated by affinity adsorption and 2-D gel electrophoresis. Incubation of the algal extracts with a 38-mer duplex oligonucleotide carrying a single DNA simple mispair generated a few gel retardation complexes. G-T mispair was recognized significantly better than C-T, G-G, G-A, and C-C mispairs by the algal extracts and these extracts bound very weakly to G-A and C-C mispairs, displaying a universal trend of mismatch binding efficiency. The levels of mismatch recognition complexes were slightly increased in the presence of 1 mM ATP. Two 13-kDa G-T binding polypeptides possessing p Is of 5.3 and 5.5 were isolated after resolving affinity-captured proteins by 2-D gel electrophoresis and the two factors were found to bind 5.5- and 2.8-fold stronger to heteroduplex than to homoduplex DNA, respectively. No proteins significantly homologous to the two algal G-T binding proteins were found by peptide mass fingerprinting (PMF). The sequence of a peptide generated from trypsin-cleavage of one G-T binding factor (p I 5.5) could be aligned with the amino acid sequences that form the C-terminal active sites of human and mouse mismatch-specific uracil/thymine-DNA glycosylases, suggesting the possibility of this factor as an algae- or a Chlorella-specific DNA mismatch glycosylase.

Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex

Cadmium (Cd2+) is a known carcinogen that inactivates the DNA mismatch repair (MMR) pathway. In this study, we have tested the effect of Cd2+ exposure on the enzymatic activity of the mismatch binding complex MSH2–MSH6. Our results indicate that Cd2+ is highly inhibitory to the ATP binding and hydrolysis activities of MSH2–MSH6, and less inhibitory to its DNA mismatch binding activity. The inhibition of the ATPase activity appears to be dose and exposure time dependent. However, the inhibition of the ATPase activity by Cd2+ is prevented by cysteine and histidine, suggesting that these residues are essential for the ATPase activity and are targeted by Cd2+. A comparison of the mechanism of inhibition with N-ethyl maleimide, a sulfhydryl group inhibitor, indicates that this inhibition does not occur through direct inactivation of sulfhydryl groups. Zinc (Zn2+) does not overcome the direct inhibitory effect of Cd2+ on the MSH2–MSH6 ATPase activity in vitro. However, the increase in the mutator phenotype of yeast cells exposed to Cd2+ was prevented by excess Zn2+, probably by blocking the entry of Cd2+ into the cell. We conclude that the inhibition of MMR by Cd2+ is through the inactivation of the ATPase activity of the MSH2–MSH6 heterodimer, resulting in a dominant negative effect and causing a mutator phenotype.

Disruption of the helix-u-turn-helix motif of MutS protein: loss of subunit dimerization, mismatch binding and ATP hydrolysis

The DNA mismatch repair protein, MutS, is a dimeric protein that recognizes mismatched bases and has an intrinsic ATPase activity. Here, a series of Taq MutS proteins having C-terminal truncations in the vicinity of a highly conserved helix-u-turn-helix (HuH) motif are assessed for subunit oligomerization, ATPase activity and DNA mismatch binding. Those proteins containing an intact HuH region are dimers; those without the HuH region are predominantly monomers in solution. Steady-state kinetics of truncated but dimeric MutS proteins reveals only modest decreases in their ATPase activity compared to full-length protein. In contrast, disruption of the HuH region results in a greatly attenuated ATPase activity. In addition, only dimeric MutS proteins are proficient for mismatch binding. Finally, an analysis of the mismatch repair competency of truncated Escherichia coli MutS proteins in a rifampicin mutator assay confirms that the HuH region is critical for in vivo function. These findings indicate that dimerization is critical for both the ATPase and DNA mismatch binding activities of MutS, and corroborate several key features of the MutS structure recently deduced from X-ray crystallographic studies.

Identification and Characterization of a Thermostable MutS Homolog from Thermus aquaticus

Recognition of mispaired or unpaired bases during DNA mismatch repair is carried out by the MutS protein family. Here, we describe the isolation and characterization of a thermostable MutS homolog from Thermus aquaticus YT-1. Sequencing of the mutS gene predicts an 89.3-kDa polypeptide sharing extensive amino acid sequence homology with MutS homologs from both prokaryotes and eukaryotes. Expression of the T. aquaticus mutS gene in Escherichia coli results in a dominant mutator phenotype. Initial biochemical characterization of the thermostable MutS protein, which was purified to apparent homogeneity, reveals two thermostable activities, an ATP hydrolysis activity in which ATP is hydrolyzed to ADP and Pi and a specific DNA mismatch binding activity with affinities for heteroduplex DNAs containing either an insertion/deletion of one base or a GT mismatch. The ATPase activity exhibits a temperature optimum of approximately 80 degrees C. Heteroduplex DNA binding by the T. aquaticus MutS protein requires Mg2+ and occurs over a broad temperature range from 0 degrees C to at least 70 degrees C. The thermostable MutS protein may be useful for further biochemical and structural studies of mismatch binding and for applications involving mutation detection.

Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage.

Acquired resistance to alkylating agents such as N-methyl-N-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine results from the ability to tolerate the potentially cytotoxic methylated base O6-methylguanine (m6-G) in DNA. In the absence of repair by demethylation in situ, m6-G is probably lethal through its inappropriate processing by the cell. DNA mismatch correction is an attractive candidate for the processing function because although it is replicated, m6-G has no perfect complementary base. Thus, m6-G in DNA might provoke abortive mismatch repair and tolerance could subsequently arise through loss of a mismatch repair pathway. Mismatch correction helps maintain genomic fidelity by removing misincorporated bases and deaminated 5-methylcytosine from DNA, and its loss by mutation confers a mutator phenotype on Escherichia coli. Here we describe human and hamster cell lines that are tolerant to N-methyl-N-nitrosourea and are defective in a DNA mismatch binding activity. The loss of this activity, which acts on G.T mispairs, confers a mutator phenotype.

Characterization of a DNA mismatch-binding activity in yeast extracts.

An activity present in nuclear extracts of the yeast Saccharomyces cerevisiae binds specifically to oligonucleotides containing DNA mismatches, as judged by a band shift assay. The specificity of this activity for mismatched DNA was confirmed by competition experiments; binding to radiolabeled heteroduplexes was abolished in the presence of excess unlabeled heteroduplex but not when excess unlabeled homoduplex was added. Both T/G and T/- (single base deletion) mispairs were recognized in each of two sequence contexts. Binding was also observed with G/G, G/A, A/C, and T/C mismatches, but recognition of a C/C mispair was very weak. Competition studies with the various mismatches were consistent with the idea that a single activity recognizes all mispairs tested. Extracts from strains mutant in either or both of two putative mismatch recognition functions, MSH2 and MSH3, were also tested. Mismatch-binding activity was present in extracts of msh3- strains but completely absent in msh2- strains. The molecular weight of the major binding protein was estimated by UV cross-linking experiments to be approximately 110 kDa, in good agreement with the size predicted for Msh2 protein (Reenan, R. A. and Kolodner, R. D. (1992) Genetics 132, 963-973).