Tyr212: A Key Residue Involved in Strand Discrimination by the DNA Mismatch Repair Endonuclease MutH

Friedhoff, Peter; Thomas, Evangelos; Pingoud, Alfred

doi:10.1016/s0022-2836(02)01224-x

Cited by 26 publications

(14 citation statements)

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“…Site-directed Mutagenesis-pTX418/Cys-free containing the gene for a cysteine-free MutL variant (termed LCys-free) was generated by replacing all seven codons for the native cysteine residues from the pTX418 template using the QuikChange protocol (Stratagene) essentially as described previously (21). The substitutions C61S, C216L, C256F, C276F, C446S, C480S, and C588S are numbered with respect to the E. coli MutL sequence.…”

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confidence: 99%

Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking

Giron-Monzon

Manelytė

Ahrends

et al. 2004

Journal of Biological Chemistry

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Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH⅐MutL complex and to explain the ATP dependence of this interaction.DNA repair systems are crucial for maintaining a stable genome (1). One of these systems, DNA mismatch repair, is present in most organisms and enhances the fidelity of DNA replication by a factor of up to 1000 by correcting replication errors (2-6). mismatch repair is a complex process that requires the coordination of a number of specialized enzymatic activities involving many different proteins. The paradigm of such a process is the MutHLS system from Escherichia coli (7). Because mismatch repair proteins MutS and MutL are evolutionarily conserved among the three kingdoms of life, it is believed that the basic mechanisms of mismatch repair are similar. In the presence of ATP and a mismatch, MutS recruits MutL and together they activate MutH, which in turn cleaves the newly replicated daughter strand at a transiently unmethylated d(GATC) site. The nick marks the erroneous strand for excision, which is started by the action of DNA helicase II, single-stranded DNA-binding protein, and one of several exonucleases (8). DNA polymerase III, single-stranded DNA-binding protein, and DNA ligase carry out repair synthesis.One of the interesting questions in DNA mismatch repair is how the different Mut proteins interact in time and space. In the E. coli system, the assembly of the active MutS⅐MutL complex will activate the strand discrimination endonuclease MutH. Both MutS and MutL are ATPases, and the roles of ATP binding and hydrolysis are just emerging (9 -13). The mismatch activated form of this complex will stimulate the latent endonuclease activity of MutH to cleave the transiently unmethylated error-containing daughter strand, thereby marking this strand for removal. We have previously shown that the proper discrimination of the unmethylated strand from the methylated strand can be mapped to a single amino acid residue in MutH, i.e. Tyr 212 . Biochemical analyses indicated that only MutL, not MutS, interacts direc...

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confidence: 99%

Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking

Giron-Monzon

Manelytė

Ahrends

et al. 2004

Journal of Biological Chemistry

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“…2 A). Finally, the resulting gap is filled in by DNA Pol III and ligated by DNA ligase I. Mutational analysis revealed that Tyr 212 of MutH is important, if not the only amino acid residue that is responsible for verification of the DNA methylation status at dam sites [15]. Methylation of both adenine residues in a Figure 2.…”

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“…Binding is verified when all contacts of the bases of the recognition sequence are properly formed. This leads to activation of the catalytic centre and subsequent cleavage of the nonmethylated nascent strand [15]. After DNA unwinding by helicase II and protection of the template strand by single-strand binding (SSB) protein, the nicked strand is degraded towards and beyond the mismatch by one out of four exonucleases [16].…”

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confidence: 99%

DNA repair nucleases

Marti

Fleck

2004

Cellular and Molecular Life Sciences (CMLS)

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Stability of DNA largely depends on accuracy of repair mechanisms, which remove structural anomalies induced by exogenous and endogenous agents or introduced by DNA metabolism, such as replication. Most repair mechanisms include nucleolytic processing of DNA, where nucleases cleave a phosphodiester bond between a deoxyribose and a phosphate residue, thereby producing 5'-terminal phosphate and 3'-terminal hydroxyl groups. Exonucleases hydrolyse nucleotides from either the 5' or 3' end of DNA, while endonucleases incise internal sites of DNA. Flap endonucleases cleave DNA flap structures at or near the junction between single-stranded and double-stranded regions. DNA nucleases play a crucial role in mismatch repair, nucleotide excision repair, base excision repair and double-strand break repair. In addition, nucleolytic repair functions are required during replication to remove misincorporated nucleotides, Okazaki fragments and 3' tails that may be formed after repair of stalled replication forks.

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“…Two isoschizomers of MboI, namely Sau3AI and MutH (a nuclease involved in mismatch repair, but related to Type II restriction enzymes), in contrast to MboI belong to the EcoRV branch; they are related to each other but not to MboI. Furthermore, whereas MutH is a monomer that nicks DNA, Sau3AI is a monomer that dimerizes on the DNA and makes a double strand cut, activated by binding to a second recognition site (like a Type IIE enzyme) (40,58). MboI and Sau3AI/MutH are therefore examples of independent invention of the same functionality (specificity toward GATC), i.e.…”

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Specificity Changes in the Evolution of Type II Restriction Endonucleases

Pingoud

Sudina

Geyer

et al. 2005

Journal of Biological Chemistry

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How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (2CCNGG), PspGI (2CCWGG), Eco-RII (2CCWGG), NgoMIV (G2CCGGC), and Cfr10I (R2CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of ϳ70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (2GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of ϳ70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.

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Tyr212: A Key Residue Involved in Strand Discrimination by the DNA Mismatch Repair Endonuclease MutH

Cited by 26 publications

References 53 publications

Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking

Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking

DNA repair nucleases

Specificity Changes in the Evolution of Type II Restriction Endonucleases

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