Interaction of MutS Protein with the Major and Minor Grooves of a Heteroduplex DNA
1997; Elsevier BV; Volume: 272; Issue: 20 Linguagem: Inglês
10.1074/jbc.272.20.13355
ISSN1083-351X
Autores Tópico(s)DNA Repair Mechanisms
ResumoThermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of TaqMutS protein to a heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24–28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish thatTaq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1,10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA. Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of TaqMutS protein to a heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24–28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish thatTaq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1,10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA. DNA mismatch repair is crucial for maintaining the integrity of the genome. This DNA repair pathway corrects mispaired or unpaired bases that arise by replication errors, by physical damage to bases (e.g. deamination of 5-methylcytosine), and by the formation of heteroduplex DNA during homologous recombination between similar but not identical sequences (reviewed in Ref. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar). In addition, mismatch repair serves to modulate levels of homologous recombination between evolutionarily divergent sequences (2Rayssiguier C. Thaler D.S. Radman M. Nature. 1989; 342: 396-401Crossref PubMed Scopus (583) Google Scholar, 3Selva E.M. New L. Crouse G.F. Lahue R.S. Genetics. 1995; 139: 1175-1188Crossref PubMed Google Scholar). More recently, mismatch repair has been implicated in transcription-coupled nucleotide excision repair and in G2 cell-cycle checkpoint control (reviewed in Ref. 4Kolodner R. Genes & Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (540) Google Scholar).Numerous genetic and biochemical studies have established the existence of mismatch repair in eukaryotes that bears overall similarity to that seen in prokaryotes including the identification of several eukaryotic MutS and MutL homologs. The recent demonstration that mutations in human MutS and MutL homologs are the underlying defect in many hereditary and sporadic tumors speaks to the prominent role this repair pathway plays in mutation avoidance (reviewed in Refs. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar, 4Kolodner R. Genes & Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (540) Google Scholar, and5Kunkel T.A. Curr. Biol. 1995; 5: 1091-1094Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).The most thoroughly studied mismatch repair system is methyl-directed mismatch repair in Escherichia coli (reviewed in Refs. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar and6Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (771) Google Scholar). E. coli MutS protein recognizes and binds to mismatches and small insertion/deletion mutations of 1–4 nucleotides. In the presence of ATP, MutL protein forms a complex with MutS bound to a heteroduplex DNA, and together they activate MutH, an endonuclease that incises the DNA at hemimethylated d(GATC) sites, thereby directing excision and repair synthesis to the newly synthesized (and transiently undermethylated) DNA strand. The nature of the strand discrimination signal in other prokaryotes and in eukaryotes where no equivalent dam methylation systems have been found is unclear, but it may involve single-strand breaks or ends (reviewed in Ref. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar).The means by which MutS proteins discriminate between damaged and undamaged DNA poses an intriguing problem in protein-DNA recognition. The mismatch repair machinery of E. coli discriminates among different mismatches (reviewed in Ref. 6Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (771) Google Scholar). G:T, A:C, A:A, and G:G mismatches are efficiently repaired, whereas the repair efficiencies of T:T, T:C, and A:G mismatches are dependent on sequence context. In contrast, C:C mismatches are very poor substrates for the mismatch repair pathway. X-ray diffraction and NMR studies indicate that, to a first approximation, most base pair mismatches are relatively well accommodated within the DNA helix and introduce only minor distortions (reviewed in Refs. 7Kennard O. J. Biomol. Struct. & Dyn. 1985; 3: 205-226Crossref PubMed Scopus (112) Google Scholar and 8Patel D.J. Shapiro L. Hare D. Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology. 1. Springer-Verlag, Berlin1987: 70-84Google Scholar). Nevertheless, these localized conformational changes can, in some cases, be detected biochemically,e.g. by alterations in the binding mode of the minor groove reporter neocarzinostatin (9Kappen L.S. Goldberg I.H. Biochemistry. 1992; 31: 9081-9089Crossref PubMed Scopus (15) Google Scholar) or by hypersensitivity to osmium tetroxide in the case of a G:T mismatch (10Bhattacharyya A. Lilley D.M.J. Nucl. Acids Res. 1989; 17: 6821-6840Crossref PubMed Scopus (159) Google Scholar). Several studies have concluded that efficient repair is, roughly speaking, correlated with the propensity for a given mismatch to be stacked within the helix. Thus, intrahelical wobble base pairs (e.g. G:T, A:C, A:A, and G:G) may be repaired more readily than looped out base pairs (e.g. C:C, C:T, and T:T) (11Fazakerley G.V. Quignard E. Woisard A. Guschlbauer W. van der Marel G.A. van B.J.H. Jones M. Radman M. EMBO J. 1986; 5: 3697-3703Crossref PubMed Scopus (91) Google Scholar, 12Su S.S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (258) Google Scholar, 13Werntges H. Steger G. Riesner D. Fritz H.-J. Nucl. Acids Res. 1986; 14: 3773-3790Crossref PubMed Scopus (149) Google Scholar). The recognition of DNA bulges is complicated by the fact that unpaired bases are, for the most part, stacked within the helix, although in some cases, unpaired pyrimidines have been observed to be extrahelical, and heteroduplexes containing unpaired bases are sometimes associated with compensatory kinks or bends (reviewed in Ref. 14Turner D.H. Curr. Opin. Struct. Biol. 1992; 2: 334-337Crossref Scopus (72) Google Scholar).To begin to address the molecular mechanism of substrate recognition by MutS protein, we have initiated biochemical studies of heteroduplex DNA binding by a thermostable MutS protein from Thermus aquaticus. This protein binds specifically to heteroduplex DNAs containing mispaired or unpaired bases over a broad temperature range up to 70 °C (15Biswas I. Hsieh P. J. Biol. Chem. 1996; 271: 5040-5048Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Here, we utilize enzymatic and chemical probes to identify regions of the heteroduplex DNA that are in close association with Taq MutS protein. Our data reveal that critical contact points reside in the immediate vicinity of an unpaired base in both the major and minor grooves of a heteroduplex DNA.DISCUSSIONEnzymatic and chemical footprinting identify regions of a heteroduplex DNA containing a single unpaired thymidine residue or a G:T mismatch that interact with a thermostable MutS mismatch repair protein. The DNase I footprint of the complex extends approximately three turns of the DNA helix more or less centered on the lesion. Chemical probes delineate protein-DNA interactions involving no more than one turn of the DNA helix in either direction flanking the lesion. Interactions involving a very limited number of nucleotides are consistent with the absence of a defined consensus sequence for mismatch recognition by MutS protein. Taq MutS protein contacts the major groove and the sugar-phosphate backbone of the heteroduplex in the region of an unpaired base. In addition, hydroxyl radical and phenanthroline-copper footprinting strongly suggest that MutS protein is also closely associated with the minor groove in the immediate vicinity of the unpaired base, although distortion of the minor groove as a consequence of binding elsewhere cannot be ruled out. Based on these data, we propose that Taq MutS makes extensive contacts with the periphery of the helix in the vicinity of the lesion.Protein contacts involving the major groove of a Δ1 insertion/deletion heteroduplex in two different sequence contexts as well as a G:T heteroduplex are identified as a result of protection from methylation of neighboring guanine residues on either side of the lesion. Methylation interference implicates the same critical guanine residues in heteroduplex binding. Two regions of hydroxyl radical protection offset in the 5′ direction are also consistent with major groove interactions in the immediate vicinity of the lesion. Independent evidence for major groove contacts involving TaqMutS protein stem from photocross-linking studies in which the cross-linking moiety is located in the major groove of a heteroduplex DNA. 1V. Malkov, I. Biswas, R. D. Camerini-Otero, and P. Hsieh, submitted for publication. Close association of the Taq MutS protein with the opposing minor groove in the vicinity of the lesion is supported by hydroxyl radical and phenanthroline-copper footprinting results. Hydroxyl radical footprinting of the Taq MutS-Δ1 heteroduplex complex reveals a pair of completely protected regions in complementary strands offset by 3 nucleotides in the 3′ direction. This pattern of strong protection from hydroxyl radical cleavage is characteristic of minor groove binding as first demonstrated for λ repressor (22Tullius T.D. Dombroski B.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 87: 5469-5473Crossref Scopus (474) Google Scholar) and subsequently for other proteins including E. coli IHF protein (32Yang C.-C. Nash H.A. Cell. 1989; 57: 869-880Abstract Full Text PDF PubMed Scopus (228) Google Scholar), TFIID (33Lee D.K. Horikoshi M. Roeder R.G. Cell. 1991; 67: 1241-1250Abstract Full Text PDF PubMed Scopus (128) Google Scholar), and Flp recombinase (17Kimball A. Kimball M.L. Jayaram M. Tullius T.D. Nucleic Acids Res. 1995; 23: 3009-3017Crossref PubMed Scopus (11) Google Scholar). Although we cannot rule out MutS-induced distortion of the minor groove in the absence of minor groove binding, the pattern of hydroxyl radical cleavage does rule out alignment of Taq MutS protein along only one side of the DNA, since no pattern of periodic protection coinciding with the helical repeat of DNA was observed.Binding of Taq MutS protein to a Δ1 or G:T heteroduplex also affords complete protection from 1,10-phenanthroline-copper modification of approximately 6 residues on both strands centered on the lesion (Figs. 6 and 8 B). Such extensive protection in conjunction with hydroxyl radical footprinting, discussed above, and ethylation interference, see below, argue strongly for protein contacts involving the minor groove.Ethylation interference studies identify several phosphates in both strands of the heteroduplex as being important for binding and are consistent with the protein contacting both the major and minor grooves in the vicinity of the unpaired base (see Fig. 10). Ethylation of phosphates located 3′ to every critical guanine residue identified in methylation protection/interference studies abolished MutS binding. Likewise, ethylation of phosphates 3′ to every protected residue identified in hydroxyl radical footprinting experiments, with the exception of the unpaired thymidine, also abolished binding.Our results confirm and extend previous studies concerning interactions between MutS proteins and heteroduplex DNA. DNase I protection and methidiumpropyl-EDTA·Fe(II) cleavage experiments of E. coli MutS protein bound to various mismatches revealed highly localized interactions (12Su S.S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (258) Google Scholar). E. coli MutS protected about 22 bases in each strand from DNase I cleavage, although, in contrast toTaq MutS protein, the pattern of protection for E. coli MutS protein was asymmetric with respect to the mismatch (insertion/deletion mutations were not examined). In the case of the methidiumpropyl-EDTA·Fe(II) footprinting, protection extended some 8–12 bases, depending on the mismatch. Methylation interference studies of partially purified human GTBP bound to a G:T mismatch identified flanking guanines and to some extent adenosines as being important for binding, although methylation of guanines 2 or more bases away from the mismatch had no effect (34Jiricny J. Hughes M. Corman N. Rudkin B.B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8860-8864Crossref PubMed Scopus (106) Google Scholar).The interaction of MutS protein with the heteroduplex DNA is reminiscent of the binding interactions of another DNA repair protein,E. coli MutY, that recognizes G:A mismatches (35Lu A.-L. Tsai-Wu J.-J. Cillo J. J. Biol. Chem. 1995; 270: 23582-23588Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Based on alkylation interference and substrate specificity studies, those authors concluded that MutY binds to both the major and minor grooves in the vicinity of the mismatch as well as to several critical phosphate groups.Central to the issue of recognition of mispaired and unpaired bases by MutS proteins is the structure of the DNA heteroduplex containing such a lesion. Unfortunately, our current knowledge of DNA structure as it relates to mismatches or bulges is far from complete. However, NMR, x-ray diffraction, electrophoretic, and thermodynamic studies reveal little structural similarity between DNA bulges and mismatches.Both x-ray crystallographic and NMR studies reveal that the G:T mismatch assumes a "wobble" type hydrogen bonding scheme utilizing the major tautomers of the bases (reviewed in Refs. 28Kennard O. Salisbury S.A. J. Biol. Chem. 1993; 268: 10701-10704Abstract Full Text PDF PubMed Google Scholar, 36Hunter W.N. Methods Enzymol. 1992; 211: 221-231Crossref PubMed Scopus (19) Google Scholar, 37Patel D.J. Shapiro L. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 423-454Crossref PubMed Scopus (95) Google Scholar). In this structure, the thymine is displaced toward the major groove. Distortions of the G:T heteroduplex are not as extensive as for a bulged DNA (see below), although the mismatch does result in localized structural distortions including alterations in base stacking in adjacent residues in some instances (8Patel D.J. Shapiro L. Hare D. Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology. 1. Springer-Verlag, Berlin1987: 70-84Google Scholar). In addition, a G:T mismatch has been shown to alter the attack site of the minor groove intercalator neocarzinostatin (9Kappen L.S. Goldberg I.H. Biochemistry. 1992; 31: 9081-9089Crossref PubMed Scopus (15) Google Scholar), and the mispaired thymidine in a G:T mismatch can be hypersensitive to modification by osmium tetroxide (10Bhattacharyya A. Lilley D.M.J. Nucl. Acids Res. 1989; 17: 6821-6840Crossref PubMed Scopus (159) Google Scholar).Unpaired bases can be extrahelical or stacked within the helix. Unpaired purines are largely stacked within the DNA helix at all temperatures and in several different sequence contexts (38Kalnik M.W. Norman D.G. Swann P.F. Patel D.J. J. Biol. Chem. 1989; 264: 3702-3712Abstract Full Text PDF PubMed Google Scholar, 39Woodson S.A. Crothers D.M. Biochemistry. 1988; 27: 3130-3141Crossref PubMed Scopus (86) Google Scholar, 40Rosen M.A. Live D. Patel D.J. Biochemistry. 1992; 31: 4004-4014Crossref PubMed Scopus (70) Google Scholar, 41Aboula-ela F. Murchie A.I.H. Homans S.W. Lilley D.M.J. J. Mol. Biol. 1993; 229: 173-188Crossref PubMed Scopus (35) Google Scholar). Pyrimidine bulges can be either extrahelical or intrahelical, depending on sequence and temperature. In general, pyrimidine bulges are looped out at lower temperatures, i.e. 0 °C, and stacked within the helix at higher temperatures, i.e. 35 °C (30Kalnik M.W. Norman D.G. Zagorski M.G. Swann P.F. Patel D.J. Biochemistry. 1989; 28: 294-303Crossref PubMed Scopus (85) Google Scholar,42Morden K.M. Chu Y.G. Martin F.H. Tinoco I. Biochemistry. 1983; 22: 5557-5563Crossref Scopus (101) Google Scholar, 43van den Hoogen Y.T. van Beuzekom A.A. van den Elst H. van der Marel G.A. van Boom J.H. Altona C. Nucleic Acids Res. 1988; 16: 2971-2986Crossref PubMed Scopus (42) Google Scholar, 44Kalnik M.W. Norman D.G. Li B.F. Swann P.F. Patel D.J. J. Biol. Chem. 1990; 265: 636-647Abstract Full Text PDF PubMed Google Scholar, 45Morden K.M. Gunn B.M. Maskos K. Biochemistry. 1990; 29: 8835-8845Crossref PubMed Scopus (56) Google Scholar). In some although not all cases, the presence of a DNA bulge confers a bend in the DNA helix (reviewed in Ref. 14Turner D.H. Curr. Opin. Struct. Biol. 1992; 2: 334-337Crossref Scopus (72) Google Scholar). The extent of bending can be influenced by the identity of the nucleotides in the bulge (unpaired purines cause greater bending than unpaired pyrimidines), the size of the bulge, and the flanking sequence (10Bhattacharyya A. Lilley D.M.J. Nucl. Acids Res. 1989; 17: 6821-6840Crossref PubMed Scopus (159) Google Scholar, 46Rice J.A. Crothers D.M. Biochemistry. 1989; 28: 4512-4516Crossref PubMed Scopus (90) Google Scholar,47Wang Y.-H. Griffith J. Biochemistry. 1991; 30: 1358-1363Crossref PubMed Scopus (58) Google Scholar). While the conformation of the unpaired thymidine in the DNA substrate shown in Fig. 2 is unknown, it may be stacked within the helix, as has been established by NMR for another bulged heteroduplex consisting of one unpaired thymidine flanked by guanine residues (44Kalnik M.W. Norman D.G. Li B.F. Swann P.F. Patel D.J. J. Biol. Chem. 1990; 265: 636-647Abstract Full Text PDF PubMed Google Scholar). If the thymidine is stacked within the helix, it is still subject to hypermodification by 1,10-phenanthroline-copper and OsO4prior to being bound by MutS, and the presence of the unpaired base introduces distortion of neighboring residues as detected by phenanthroline-copper footprinting of naked heteroduplexes.A possible mechanism for the recognition of insertion/deletion and mismatch lesions by MutS proteins is one that has been invoked for nucleotide excision repair to explain damage-specific recognition of a broad spectrum of DNA lesions that share no obvious structural similarities (reviewed in Refs. 48Hanawalt P.C. Mutat. Res. 1993; 289: 7-15Crossref PubMed Scopus (11) Google Scholar and 49Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar)). In this scheme, recognition is mediated through protein-induced deformation of the DNA rather than direct recognition of structurally diverse substrates. Presumably, lesions that are targets for excision repair facilitate the formation of an altered conformation, thereby shifting the equilibrium in favor of the formation of a stable protein-DNA complex. Based on the work presented here and the work of others, we speculate thatTaq MutS binds and distorts the heteroduplex DNA and utilizes extensive contacts with the periphery of the helix to gauge the extent to which a region of DNA can accommodate protein-induced strain. Both Mg2+ and ATP cofactors have been shown to modulate heteroduplex binding by MutS proteins (reviewed in Refs. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar and4Kolodner R. Genes & Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (540) Google Scholar), and such modulation may be an integral component of the target search by MutS. In this regard, it is noteworthy that E. coli MutS protein has been shown to form α structures in the presence of ATP, suggesting that it can translocate along the DNA (6Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (771) Google Scholar).This study provides new details on the interactions between MutS protein and a heteroduplex DNA. Problems to be pursued include the identification of regions of MutS protein that contact the major and minor grooves of the heteroduplex, the determination of protein-induced DNA distortion, and an examination of how contacts between MutS protein and the heteroduplex DNA are modulated during repair. DNA mismatch repair is crucial for maintaining the integrity of the genome. This DNA repair pathway corrects mispaired or unpaired bases that arise by replication errors, by physical damage to bases (e.g. deamination of 5-methylcytosine), and by the formation of heteroduplex DNA during homologous recombination between similar but not identical sequences (reviewed in Ref. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar). In addition, mismatch repair serves to modulate levels of homologous recombination between evolutionarily divergent sequences (2Rayssiguier C. Thaler D.S. Radman M. Nature. 1989; 342: 396-401Crossref PubMed Scopus (583) Google Scholar, 3Selva E.M. New L. Crouse G.F. Lahue R.S. Genetics. 1995; 139: 1175-1188Crossref PubMed Google Scholar). More recently, mismatch repair has been implicated in transcription-coupled nucleotide excision repair and in G2 cell-cycle checkpoint control (reviewed in Ref. 4Kolodner R. Genes & Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (540) Google Scholar). Numerous genetic and biochemical studies have established the existence of mismatch repair in eukaryotes that bears overall similarity to that seen in prokaryotes including the identification of several eukaryotic MutS and MutL homologs. The recent demonstration that mutations in human MutS and MutL homologs are the underlying defect in many hereditary and sporadic tumors speaks to the prominent role this repair pathway plays in mutation avoidance (reviewed in Refs. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar, 4Kolodner R. Genes & Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (540) Google Scholar, and5Kunkel T.A. Curr. Biol. 1995; 5: 1091-1094Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The most thoroughly studied mismatch repair system is methyl-directed mismatch repair in Escherichia coli (reviewed in Refs. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar and6Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (771) Google Scholar). E. coli MutS protein recognizes and binds to mismatches and small insertion/deletion mutations of 1–4 nucleotides. In the presence of ATP, MutL protein forms a complex with MutS bound to a heteroduplex DNA, and together they activate MutH, an endonuclease that incises the DNA at hemimethylated d(GATC) sites, thereby directing excision and repair synthesis to the newly synthesized (and transiently undermethylated) DNA strand. The nature of the strand discrimination signal in other prokaryotes and in eukaryotes where no equivalent dam methylation systems have been found is unclear, but it may involve single-strand breaks or ends (reviewed in Ref. 1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar). The means by which MutS proteins discriminate between damaged and undamaged DNA poses an intriguing problem in protein-DNA recognition. The mismatch repair machinery of E. coli discriminates among different mismatches (reviewed in Ref. 6Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (771) Google Scholar). G:T, A:C, A:A, and G:G mismatches are efficiently repaired, whereas the repair efficiencies of T:T, T:C, and A:G mismatches are dependent on sequence context. In contrast, C:C mismatches are very poor substrates for the mismatch repair pathway. X-ray diffraction and NMR studies indicate that, to a first approximation, most base pair mismatches are relatively well accommodated within the DNA helix and introduce only minor distortions (reviewed in Refs. 7Kennard O. J. Biomol. Struct. & Dyn. 1985; 3: 205-226Crossref PubMed Scopus (112) Google Scholar and 8Patel D.J. Shapiro L. Hare D. Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology. 1. Springer-Verlag, Berlin1987: 70-84Google Scholar). Nevertheless, these localized conformational changes can, in some cases, be detected biochemically,e.g. by alterations in the binding mode of the minor groove reporter neocarzinostatin (9Kappen L.S. Goldberg I.H. Biochemistry. 1992; 31: 9081-9089Crossref PubMed Scopus (15) Google Scholar) or by hypersensitivity to osmium tetroxide in the case of a G:T mismatch (10Bhattacharyya A. Lilley D.M.J. Nucl. Acids Res. 1989; 17: 6821-6840Crossref PubMed Scopus (159) Google Scholar). Several studies have concluded that efficient repair is, roughly speaking, correlated with the propensity for a given mismatch to be stacked within the helix. Thus, intrahelical wobble base pairs (e.g. G:T, A:C, A:A, and G:G) may be repaired more readily than looped out base pairs (e.g. C:C, C:T, and T:T) (11Fazakerley G.V. Quignard E. Woisard A. Guschlbauer W. van der Marel G.A. van B.J.H. Jones M. Radman M. EMBO J. 1986; 5: 3697-3703Crossref PubMed Scopus (91) Google Scholar, 12Su S.S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (258) Google Scholar, 13Werntges H. Steger G. Riesner D. Fritz H.-J. Nucl. Acids Res. 1986; 14: 3773-3790Crossref PubMed Scopus (149) Google Scholar). The recognition of DNA bulges is complicated by the fact that unpaired bases are, for the most part, stacked within the helix, although in some cases, unpaired pyrimidines have been observed to be extrahelical, and heteroduplexes containing unpaired bases are sometimes associated with compensatory kinks or bends (reviewed in Ref. 14Turner D.H. Curr. Opin. Struct. Biol. 1992; 2: 334-337Crossref Scopus (72) Google Scholar). To begin to address the molecular mechanism of substrate recognition by MutS protein, we have initiated biochemical studies of heteroduplex DNA binding by a thermostable MutS protein from Thermus aquaticus. This protein binds specifically to heteroduplex DNAs containing mispaired or unpaired bases over a broad temperature range up to 70 °C (15Biswas I. Hsieh P. J. Biol. Chem. 1996; 271: 5040-5048Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Here, we utilize enzymatic and chemical probes to identify regions of the heteroduplex DNA that are in close association with Taq MutS protein. Our data reveal that critical contact points reside in the immediate vicinity of an unpaired base in both the major and minor grooves of a heteroduplex DNA. DISCUSSIONEnzymatic and chemical footprinting identify regions of a heteroduplex DNA containing a single unpaired thymidine residue or a G:T mismatch that interact with a thermostable MutS mismatch repair protein. The DNase I footprint of the complex extends approximately three turns of the DNA helix more or less centered on the lesion. Chemical probes delineate protein-DNA interactions involving no more than one turn of the DNA helix in either direction flanking the lesion. Interactions involving a very limited number of nucleotides are consistent with the absence of a defined consensus sequence for mismatch recognition by MutS protein. Taq MutS protein contacts the major groove and the sugar-phosphate backbone of the heteroduplex in the region of an unpaired base. In addition, hydroxyl radical and phenanthroline-copper footprinting strongly suggest that MutS protein is also closely associated with the minor groove in the immediate vicinity of the unpaired base, although distortion of the minor groove as a consequence of binding elsewhere cannot be ruled out. Based on these data, we propose that Taq MutS makes extensive contacts with the periphery of the helix in the vicinity of the lesion.Protein contacts involving the major groove of a Δ1 insertion/deletion heteroduplex in two different sequence contexts as well as a G:T heteroduplex are identified as a result of protection from methylation of neighboring guanine residues on either side of the lesion. Methylation interference implicates the same critical guanine residues in heteroduplex binding. Two regions of hydroxyl radical protection offset in the 5′ direction are also consistent with major groove interactions in the immediate vicinity of the lesion. Independent evidence for major groove contacts involving TaqMutS protein stem from photocross-linking studies in which the cross-linking moiety is located in the major groove of a heteroduplex DNA. 1V. Malkov, I. Biswas, R. D. Camerini-Otero, and P. Hsieh, submitted for publication. Close association of the Taq MutS protein with the opposing minor groove in
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