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Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding.

ABSTRACT: Previous structural studies on native T5 5' nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was proposed in which single-stranded DNA threads through the archway. We investigated the kinetic and substrate-binding characteristics of wild-type and mutant nucleases in relation to the proposed model. Five basic residues R33, K215, K241, R172, and R216, are all implicated in binding branched DNA substrates. All these residues except R172 are involved in binding to duplex DNA carrying a 5' overhang. Replacement of either K215 or R216 with a neutral amino acid did not alter kcat appreciably. However, these mutant nucleases displayed significantly increased values for Kd and Km. A comparison of flap endonuclease binding to pseudoY substrates and duplexes with a single-stranded 5' overhang suggests a better model for 5' nuclease-DNA binding. We propose a major revision to the binding model consistent with these biophysical data.

SUBMITTER: Dervan JJ

PROVIDER: S-EPMC124304 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding.

Dervan Joe J JJ Feng Min M Patel Dipak D Grasby Jane A JA Artymiuk Peter J PJ Ceska Thomas A TA Sayers Jon R JR

Proceedings of the National Academy of Sciences of the United States of America 20020601 13

Previous structural studies on native T5 5' nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was proposed in which single-stranded DNA threads through the archway. We investigated the kinetic and substrate-binding characteristics of wild-type and ...[more]

PMID: 12084915

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Project description:Ras oncogenes (Hras, Kras and Nras) are important drivers of carcinogenesis. However, tumors with Ras mutations often show loss of the corresponding wild-type (WT) allele, suggesting that proto-oncogenic forms of Ras can function as a suppressor of carcinogenesis. In vitro studies also suggest that WT Ras proteins can suppress the tumorigenic properties of alternate mutant Ras family members, but in vivo evidence for these heterologous interactions is lacking. We have investigated the genetic interactions between different combinations of mutant and WT Ras alleles in vivo using carcinogen-induced lung and skin carcinogenesis in mice with targeted deletion of different Ras family members. The major suppressor effect of WT Kras is observed only in mutant Kras-driven lung carcinogenesis, where loss of one Kras allele led to increased tumor number and size. Deletion of one Hras allele dramatically reduced the number of skin papillomas with Hras mutations, consistent with Hras as the major target of mutation in these tumors. However, skin carcinoma numbers were very similar, suggesting that WT Hras functions as a suppressor of progression from papillomas to invasive squamous carcinomas. In the skin, the Kras proto-oncogene functions cooperatively with mutant Hras to promote papilloma development, although the effect is relatively small. In contrast, the Hras proto-oncogene attenuated the activity of mutant Kras in lung carcinogenesis. Interestingly, loss of Nras increased the number of mutant Kras-induced lung tumors, but decreased the number of mutant Hras-induced skin papillomas. These results show that the strongest suppressor effects of WT Ras are only seen in the context of mutation of the cognate Ras protein, and only relatively weak effects are detected on tumor development induced by mutations in alternative family members. The data also underscore the complex and context-dependent nature of interactions between proto-oncogenic and oncogenic forms of different Ras family members during tumor development.

Dataset Information

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding.

Publications

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding.

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