A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac+ revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We report that expression of wild-type hMLH1 protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA. Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins.
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April 2003
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Research Article|
April 01 2003
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
Barbara QUARESIMA;
Barbara QUARESIMA
∗Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Università degli Studi di Catanzaro ‘Magna Græcia’, Via Tommaso Campanella 115, 88100 Catanzaro, Italy
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Pietro ALIFANO;
Pietro ALIFANO
†Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università degli Studi di Lecce, Via Monteroni, 73100 Lecce, Italy,
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Pierfrancesco TASSONE;
Pierfrancesco TASSONE
∗Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Università degli Studi di Catanzaro ‘Magna Græcia’, Via Tommaso Campanella 115, 88100 Catanzaro, Italy
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Enrico V. AVVEDIMENTO;
Enrico V. AVVEDIMENTO
‡Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘L. Califano’, Università degli Studi di Napoli ‘Federico II’, Via S. Pansini 5, 80131 Napoli, Italy
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Francesco S. COSTANZO;
Francesco S. COSTANZO
1
∗Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Università degli Studi di Catanzaro ‘Magna Græcia’, Via Tommaso Campanella 115, 88100 Catanzaro, Italy
1To whom correspondence should be addressed (e-mail fsc@unicz.it).
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Salvatore VENUTA
Salvatore VENUTA
∗Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Università degli Studi di Catanzaro ‘Magna Græcia’, Via Tommaso Campanella 115, 88100 Catanzaro, Italy
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Publisher: Portland Press Ltd
Received:
July 31 2002
Revision Received:
December 09 2002
Accepted:
January 03 2003
Accepted Manuscript online:
January 03 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 371 (1): 183–189.
Article history
Received:
July 31 2002
Revision Received:
December 09 2002
Accepted:
January 03 2003
Accepted Manuscript online:
January 03 2003
Citation
Barbara QUARESIMA, Pietro ALIFANO, Pierfrancesco TASSONE, Enrico V. AVVEDIMENTO, Francesco S. COSTANZO, Salvatore VENUTA; 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. Biochem J 1 April 2003; 371 (1): 183–189. doi: https://doi.org/10.1042/bj20021205
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