Methyl-directed mismatch repair is bidirectional

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/1993; 268(16):11823-9.
Source: PubMed


Methyl-directed mismatch repair is initiated by the mismatch-provoked, MutHLS-dependent cleavage of the unmodified strand at a hemimethylated d(GATC) sequence. This reaction is independent of the polarity of the unmodified strand and can occur either 3' or 5' to the mismatch on the unmethylated strand (Au, K. G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148). The overall repair reaction also occurs without regard to polarity of the unmethylated strand. Both hemimethylated configurations of a linear heteroduplex containing a single d(GATC) sequence are subject to methyl-directed correction in Escherichia coli extracts and in a purified repair system. Repair of both heteroduplex orientations requires MutH, MutL, MutS, DNA helicase II, SSB, and DNA polymerase III holoenzyme, but the two substrates differ with respect to exonuclease requirements for correction. When the unmethylated d(GATC) sequence that directs repair is located 5' to the mismatch on the unmodified strand, mismatch correction requires the 5'--> 3' hydrolytic activity of exonuclease VII or RecJ exonuclease. Repair directed by an unmodified d(GATC) sequence situated 3' to the mismatch depends on the 3'--> 5' activity of exonuclease I. Specific requirements for these activities are evident with circular heteroduplexes containing a single asymmetrically placed d(GATC) sequence, with the requirement for a 5'--> 3' or 3'--> 5' hydrolytic activity being determined by the orientation of the unmethylated strand along the shorter path joining the two sites in the DNA circle. This observation suggests that the methyl-directed repair system utilizes the proximal d(GATC) sequence to direct correction. To our knowledge, these experiments represent the first instance in which exonuclease I, exonuclease VII, and RecJ have been implicated in a particular DNA metabolic pathway.

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    • "The MutH endonuclease scission was found to direct unwinding and degradation of the unmethylated DNA strand by the coordinated action of Helicase II (UvrD) and one of four single-stranded DNA (ssDNA) exonucleases (RecJ, ExoI, ExoVII, ExoX) (Matson, 1986; Viswanathan and Lovett, 1998; Yamaguchi et al., 1998). Depending on the relative location of the MutH endonuclease in-scission to the mismatch, the resulting excision gap may occur 5′→3′ or 3′→5′ but invariably traverses only the interval between a Dam-site (nick) to just past the mismatch (Figure 2) (Cooper et al., 1993; Grilley et al., 1993). "
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    ABSTRACT: The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.
    British Journal of Pharmacology 09/2009; 158(3):679-92. DOI:10.1111/j.1476-5381.2009.00423.x · 4.84 Impact Factor
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    • "It has been proposed that these four nucleases (ExoI, RecJ, ExoVII and ExoX) serve redundant functions in MMR. However, these enzymes are dissimilar and possess distinct mechanisms; their individual deletions affect other features of DNA repair in different ways (8–16) suggesting that they are not maintained in genomes as ‘backup’ activities for MMR. "
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    ABSTRACT: Exonuclease VII was first identified in 1974 as a DNA exonuclease that did not require any divalent cations for activity. Indeed, Escherichia coli ExoVII was identified in partially purified extracts in the presence of EDTA. ExoVII is comprised of two subunits (XseA and XseB) that are highly conserved and present in most sequenced prokaryotic genomes, but are not seen in eukaryotes. To better understand this exonuclease family, we have characterized an ExoVII homolog from Thermotoga maritima. Thermotoga maritima XseA/B homologs TM1768 and TM1769 were co-expressed and purified, and show robust nuclease activity at 80 degrees C. This activity is magnesium dependent and is inhibited by phosphate ions, which distinguish it from E. coli ExoVII. Nevertheless, both E. coli and T. maritima ExoVII share a similar putative active site motif with two conserved aspartate residues in the large (XseA/TM1768) subunit. We show that these residues, Asp235 and Asp240, are essential for the nuclease activity of T. maritima ExoVII. We hypothesize that the ExoVII family of nucleases can be sub-divided into two sub-families based on EDTA resistance and that T. maritima ExoVII is the first member of the branch that is characterized by EDTA sensitivity and inhibition by phosphate.
    Nucleic Acids Research 10/2008; 36(18):5992-6003. DOI:10.1093/nar/gkn588 · 9.11 Impact Factor
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    • "The resulting nick serves as the point of entry for MutL-activated UvrD helicase, which unwinds DNA double helix from the nick to about 100 nucleotides past the mismatch, and single-stranded DNA binding (SSB) protein, which stabilizes the single-stranded gap (Lahue et al., 1989). After unwinding the ssDNA flap is degraded in the 5'→3' direction by ExoVII or RecJ exonuclease, if the incision occurred 5' to the mismatch, or in the 3'→ 5' direction by ExoI, ExoVII or Exo X exonuclease, if the incision occurred 3' to the mismatch (Cooper et al., 1993; Grilley et al., 1993; Burdett et al., 2001). Finally , the SSB-stabilized single-stranded gap is filled in by DNA polymerase III holoenzyme and DNA ends are sealed by LigI. "
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    ABSTRACT: Since the discovery of the first E. coli mutator gene, mutT, most of the mutations inducing elevated spontaneous mutation rates could be clearly attributed to defects in DNA repair. MutT turned out to be a pyrophosphohydrolase hydrolyzing 8-oxodGTP, thus preventing its incorporation into DNA and suppresing the occurrence of spontaneous AT-->CG transversions. Most of the bacterial mutator genes appeared to be evolutionarily conserved, and scientists were continuously searching for contribution of DNA repair deficiency in human diseases, especially carcinogenesis. Yet a human MutT homologue--hMTH1 protein--was found to be overexpressed rather than inactivated in many human diseases, including cancer. The interest in DNA repair contribution to human diseases exploded with the observation that germline mutations in mismatch repair (MMR) genes predispose to hereditary non-polyposis colorectal cancer (HNPCC). Despite our continuously growing knowledge about DNA repair we still do not fully understand how the mutator phenotype contributes to specific forms of human diseases.
    Acta biochimica Polonica 02/2007; 54(3):435-57. · 1.15 Impact Factor
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