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• W2067215249 abstract "ATP hydrolysis by bacterial and eukaryotic MutS activities is required for their function in mismatch correction, and two different models for the role of ATP in MutS function have been proposed. In the translocation model, based on study of bacterial MutS, ATP binding reduces affinity of the protein for a mismatch and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997)EMBO J. 16, 4467–4476). The molecular switch model, based on study of human MutSα, invokes mismatch recognition by the MutSα·ADP complex. After recruitment of downstream repair activities to the MutSα·mismatch complex, ATP binding results in release of MutSα from the heteroduplex (Gradia, S., Acharya, S., and Fishel, R.(1997) Cell 91, 995–1005). To further clarify the function of ATP binding and hydrolysis in human MutSα action, we evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of protein·DNA complexes. All of these nucleotides were found to increase the rate of dissociation of MutSα from oligonucleotide heteroduplexes. These experiments also showed that ADP is not required for mismatch recognition by MutSα, but that the nucleotide alters the dynamics of formation and dissociation of specific complexes. Analysis of the mechanism of ATP-promoted dissociation of MutSα from a 200-base pair heteroduplex demonstrated that dissociation occurs at DNA ends in a reaction dependent on ATP hydrolysis, implying that release from this molecule involves movement of the protein along the helix contour as predicted for a translocation mechanism. In order to reconcile the relatively large rate of movement of MutS homologs along the helix with their modest rate of ATP hydrolysis, we propose a novel mechanism for protein translocation along DNA that supports directional movement over long distances with minimal energy input. ATP hydrolysis by bacterial and eukaryotic MutS activities is required for their function in mismatch correction, and two different models for the role of ATP in MutS function have been proposed. In the translocation model, based on study of bacterial MutS, ATP binding reduces affinity of the protein for a mismatch and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997)EMBO J. 16, 4467–4476). The molecular switch model, based on study of human MutSα, invokes mismatch recognition by the MutSα·ADP complex. After recruitment of downstream repair activities to the MutSα·mismatch complex, ATP binding results in release of MutSα from the heteroduplex (Gradia, S., Acharya, S., and Fishel, R.(1997) Cell 91, 995–1005). To further clarify the function of ATP binding and hydrolysis in human MutSα action, we evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of protein·DNA complexes. All of these nucleotides were found to increase the rate of dissociation of MutSα from oligonucleotide heteroduplexes. These experiments also showed that ADP is not required for mismatch recognition by MutSα, but that the nucleotide alters the dynamics of formation and dissociation of specific complexes. Analysis of the mechanism of ATP-promoted dissociation of MutSα from a 200-base pair heteroduplex demonstrated that dissociation occurs at DNA ends in a reaction dependent on ATP hydrolysis, implying that release from this molecule involves movement of the protein along the helix contour as predicted for a translocation mechanism. In order to reconcile the relatively large rate of movement of MutS homologs along the helix with their modest rate of ATP hydrolysis, we propose a novel mechanism for protein translocation along DNA that supports directional movement over long distances with minimal energy input. polymerase chain reaction base pair(s) adenosine 5′-(β,γ-imino)triphosphate adenosine 5′-O-(thiotriphosphate). In addition to their mismatch recognition activities, bacterial and eukaryotic MutS activities have an associated ATPase activity that is required for function of the proteins in mismatch repair (1Haber L.T. Walker G.C. EMBO J. 1991; 10: 2707-2715Crossref PubMed Scopus (134) Google Scholar, 2Au K.G. Welsh K. Modrich P. J. Biol. Chem. 1992; 267: 12142-12148Abstract Full Text PDF PubMed Google Scholar, 3Wu T.H. Marinus M.G. J. Bacteriol. 1994; 176: 5393-5400Crossref PubMed Google Scholar, 4Alani E. Sokolsky T. Studamire B. Miret J.J. Lahue R.S. Mol. Cell. Biol. 1997; 17: 2436-2447Crossref PubMed Scopus (111) Google Scholar, 5Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (145) Google Scholar). Two distinct functions have been proposed for nucleotide binding and hydrolysis by MutS homologs, both of which are based on the effects of ATP on MutS-heteroduplex interaction. The presence of ATP greatly reduces the efficiency of specific complex formation between bacterial MutS or eukaryotic MutSα and heteroduplex DNA (5Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (145) Google Scholar, 6Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar, 7Drummond J.T. Li G.-M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (538) Google Scholar, 8Iaccarino I. Palombo F. Drummond J. Totty N.F. Hsuan J.J. Modrich P. Jiricny J. Curr. Biol. 1996; 6: 484-486Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 9Alani E. Mol. Cell. Biol. 1996; 16: 5604-5615Crossref PubMed Scopus (142) Google Scholar, 10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), and ATP challenge of preformed MutS·heteroduplex complexes has been shown to result in departure of the protein from the mismatch (11Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar). Available information indicates that some of these effects are attributable to ATP binding. Thus, ATPγS has been shown to promote departure of MutS from the mismatch in heteroduplex DNA (11Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar), while ATPγS or ATP (in the absence of a divalent cation) reduce the binding efficiency human MutSα (hMutSα) to synthetic heteroduplexes (5Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (145) Google Scholar, 10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Electron microscopy of complexes between bacterial MutS and large heteroduplexes prepared from natural DNAs has demonstrated that ATP-promoted release of MutS from a mismatch is associated with efficient conversion of protein·DNA complexes to α-shaped loop structures stabilized by MutS at the base (11Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar). Loop formation requires a mismatch, loop size increases linearly with time, loop growth depends on continued ATP hydrolysis, and the mismatch usually ends up in the loop. These observations have been interpreted in terms of a mechanism in which ATP binding reduces affinity of the protein for a mispair and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (11Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar). MutS movement in this manner has been postulated to be important for the coupling of mismatch recognition to loading of the excision system at the strand break that directs repair (12Yamaguchi M. Dao V. Modrich P. J. Biol. Chem. 1998; 273: 9197-9201Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 13Dao V. Modrich P. J. Biol. Chem. 1998; 273: 9202-9207Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), a site that can be located hundreds of base pairs from this mismatch. The finding that ATP binding reduces the efficiency of specific complex formation between hMutSα and oligonucleotide heteroduplexes has led to proposal of a molecular switch model for action of MutS activities. Like a G-protein, hMutSα is postulated to exist in two states, an ADP-bound form that binds with near irreversible affinity to a mismatch and an ATP-bound form that does not (10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). In this proposal hMutSα·ADP binds to a mispair and recruits downstream activities to this site. After assembly of the excision system, ATP binding results in dissociation of hMutSα from the heteroduplex so that repair may proceed (10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). To further clarify the role(s) of ATP binding and hydrolysis in hMutSα action, we have evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of hMutSα·DNA complexes and have examined the effect of DNA topology on ATP-promoted dissociation of hMutSα complexes with small heteroduplexes. We demonstrate that ADP is not required for mismatch recognition by hMutSα, but that the nucleotide alters the dynamics of formation and dissociation of specific hMutSα·mismatch complexes. We also show that ATP-promoted dissociation of hMutSα from small heteroduplexes is blocked by physical barriers placed at the ends of the DNA. This implies that ATP-promoted dissociation of hMutSα from small heteroduplexes involves movement along the helix contour, as predicted for a translocation mechanism. Human MutSα was purified as described previously to a purity in excess of 95% (7Drummond J.T. Li G.-M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (538) Google Scholar). ADP and ATP levels in purified hMutSα were determined using an ATP bioluminescent assay kit (Sigma). For ATP determination, 50 μl of hMutSα (108 nm in 5 mm Tris-HCl, pH 8.0, and 1 mm EDTA) was heat-denatured at 90 °C for 5 min, followed by addition of 50 μl of 120 mm Tris acetate, pH 7.8, 20 mmMgCl2, 2 mm KCl, 3 mm EDTA, 5 mm 2-mercaptoethanol. The diluted solution was mixed with 100 μl of a 25-fold dilution of the luminescent assay mix (Sigma), placed in a scintillation vial, and photon emission determined in the tritium channel of a Beckman LS6500 scintillation counter. ATP standards prepared in a similar manner yielded a linear response throughout the range tested (0.4–12.5 nm). ADP was determined by the same procedure after conversion to ATP by pyruvate kinase (14Senior A.E. Lee R.S. al-Shawi M.K. Weber J. Arch. Biochem. Biophys. 1992; 297: 340-344Crossref PubMed Scopus (46) Google Scholar, 15Hampp R. Bergmeyer H.U. Methods of Enzymatic Analysis. 3rd Ed. 7. VCH Publishers, Inc, Deerfield Beach, FL1984: 370-379Google Scholar). Preparations of hMutSα were depleted of nucleotide using Sephadex G50 spin column chromatography. Protein samples were either kept on ice or spun through a 1-ml G50 Sephadex column equilibrated in 20 mm Tris-HCl, pH 7.6, 1 mm dithiothreitol, 5 mm MgCl2, and 50 mm KCl. Samples (50 μl) of 215 nm hMutSα, 20 mm Tris-HCl, pH 7.6, 1 mm dithiothreitol, 5 mmMgCl2, 50 mm KCl, 2 mg/ml bovine serum albumin were loaded onto spin columns, which were centrifuged briefly in a clinical centrifuge. Presence of nucleotide in gel-filtered samples was determined as described above. Oligonucleotides were purchased from Oligos Etc. (Wilsonville, OR) and, when indicated, were 5′-end labeled with T4 polynucleotide kinase (U. S. Biochemical Corp.) and [γ-32P]ATP (3000 Ci/mmol, NEN Life Science Products) to a specific activity of 1 × 106 to 3 × 106 cpm/pmol. Duplexes were prepared by mixing molar equivalents of an unlabeled or 5′-32P-labeled oligonucleotide with an appropriate unlabeled complementary sequence in 10 mm Tris-HCl, pH 8.0, 1 mm EDTA and heating to 99 °C in a Perkin-Elmer Gene Amp 9600 thermocycler, followed by cooling to 25 °C over a 30-min period. The solution of annealed duplex (50 μl) was adjusted to 1 m in NaCl and mixed with 5–10 μl of benzoylated naphthoylated DEAE-cellulose suspension (50% settled volume) in batch to remove single-stranded DNA (16Gamper H. Lehman N. Piette J. Hearst J.E. DNA. 1985; 4: 157-164Crossref PubMed Scopus (16) Google Scholar). The oligonucleotide/ benzoylated naphthoylated DEAE-cellulose mixture was layered on top of a Sephadex G50 spin column equilibrated with 10 mm Tris-HCl, pH 8.0, 1 mm EDTA, and oligonucleotide duplexes recovered after centrifugation. DNA concentration was determined by A 260 or estimated from specific radioactivity. PCR1-generated substrates were constructed by hybridizing appropriate single strands isolated from duplex DNA fragments produced by amplification of base pairs 5531–5732 of bacteriophages f1MR1 and f1MR3. The DNA sequences of the two phages are identical except for an A·T to G·C substitution at position 5632 (17Su S.-S. Lahue R.S. Au K.G. Modrich P. J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar). Forward (5′-TACGCGCAGCGTGACCGCTA) and reverse (5′-AAGTTTTTTGGGGTCGAGGT) primers were synthesized with or without a 5′-biotin tag as indicated. A smaller fragment amplified from region 5535–5732 was also prepared using the forward primer 5′-CGCAGCGTGACCGCTACACT and the reverse primer above. PCR reactions (1 ml) contained 20 mm Tris-HCl, pH 8.8, 10 mmKCl, 10 mm (NH4)2SO4, 2 mm MgSO4, 0.1% Triton X-100, 500 ng of f1MR1 or f1MR3 DNA, 1 μm of each primer, 0.2 mmdCTP, dGTP, dATP, and dTTP, and 40 units of Vent DNA polymerase (New England Biolabs). The 1-ml reaction was divided into 10 100-μl aliquots and amplified for 25 cycles using a Perkin-Elmer Gene Amp 9600 thermocycler. Incubations were at 94 °C for 15 s, 66 °C for 15 s, and 70 °C for 10 s, except that incubation time for the first stage of the first cycle was 2 min and the third stage of the 25th cycle was 2 min. The 10 aliquots were pooled and DNA product purified using a PCR purification kit (Qiagen) following the manufacturer’s protocol. The combined reactions yielded approximately 5 μg of amplified product. Single strands were isolated from PCR products by high performance liquid chromatography using a Gen-Pak Fax column (4.6 × 100 mm, Waters) under denaturing conditions. Each PCR product (∼5 μg in 100 μl) was diluted to 400 μl with buffer A (250 mmTris-HCl, 1 mm EDTA adjusted to pH 12.4 with NaOH) containing 0.5 m NaCl and injected onto the column equilibrated with this buffer at a flow rate of 0.55 ml/min. After washing for 5 min with buffer A containing 0.5 m NaCl, DNA was eluted with a gradient of NaCl (0.5–0.85 m NaCl) in buffer A over a 35-min period. The two complementary strands eluted in two distinct peaks, with elution of the first beginning at 0.65m NaCl. Under these conditions, the viral strand sequence eluted first followed by the complementary strand sequence, each recovered in a volume of about 0.5 ml. Eluted single strands were supplemented with 50 μl of 3 m sodium acetate, pH 5.2, and DNA precipitated with ethanol. Final yield was about 2 μg of each strand. 5′-Biotinylated viral strand sequence (coordinates 5531–5732) derived from of f1MR1 was combined with 5′-biotinylated complementary strand sequence (coordinates 5535–5732) prepared from f1MR1 or f1MR3 to form a homoduplex (f1MR1/f1MR1) or a G-T heteroduplex (f1MR1/MR3), respectively. Strands were annealed and the resulting duplex purified from excess single strands as described above. Resulting 201-residue fragments were biotinylated at opposing 5′-ends and contained a recessed 3′-terminus (positions 5531–5534). DNAs were end-labeled at the recessed terminus in reactions (100 μl) containing 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 50 μg/ml bovine serum albumin, 33 nm (molecules) of oligonucleotide duplex, 5 units of exo-free Klenow DNA polymerase I (Amersham Pharmacia Biotech), 25 μm dATP, dTTP, and dGTP, and 100 μCi of [α-32P]dCTP (NEN Life Science Products, 3000 Ci/mmol). Reactions were incubated at 37 °C for 5 min with radiolabeled dCTP, followed by another 10-min incubation after addition of the unlabeled triphosphates. Reactions were terminated by addition of EDTA to 24 mm, and free nucleotides were removed by G50 spin column chromatography. DNA was extracted with phenol/chloroform, precipitated with ethanol, and resuspended in 100 μl of 10 mmTris-HCl, pH 8.0, 1 mm EDTA. A 32P-labeled control 201-bp DNA that was biotin-tagged at only one end was prepared by a similar procedure, except that only one PCR primer was 5′-biotin-tagged. Binding of bacterial MutS to f1MR heteroduplex DNA containing a mismatch at position 5632 blocks cleavage byNheI endonuclease, which recognizes base pairs 5621–5626 (11Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar). We have used this assay to score hMutSα binding to a 50-bp synthetic oligonucleotide G-T heteroduplex corresponding in sequence to positions 5611–5660 of the f1MR1/f1MR3 G-T heteroduplex. Reactions (20 μl) contained 20 mm Tris-HCl, pH 7.6, 1 mmdithiothreitol, 50 μg/ml bovine serum albumin, 5 mmMgCl2, and 50 mm KCl, and [32P]G-T heteroduplex and hMutSα as indicated. After 15 min at 37 °C, 2 units of NheI (New England Biolabs) in 2.5 μl of reaction buffer were added and incubation continued for an additional 30 s. Reactions were terminated by addition of 15 μl of 40% (w/w) urea in formamide containing 0.05% xylene cyanol and 0.05% bromphenol blue. DNA fragments were separated by electrophoresis through a 10% denaturing polyacrylamide gel in 89 mm Tris, 89 mm boric acid, 2 mm EDTA, 7 murea at 40 mA. DNA species were quantitated using a Molecular Dynamics PhosphorImager. Nucleotide-promoted dissociation of hMutSα from the G-T heteroduplex was scored in a similar fashion except that preincubation with hMutSα was for 10 min, and indicated nucleotides were added together withNheI endonuclease, with subsequent incubation for 5 min. Binding reactions (20 μl) contained 20 mm Tris-HCl, pH 7.6, 1 mm dithiothreitol, 50 μg/ml bovine serum albumin, 5 mm MgCl2, 50 mm KCl, with hMutSα, 32P-labeled substrate, and unlabeled competitor DNAs present as indicated. After incubation at 37 °C for 15 min, reactions were stopped by adding 2.2 μl of 50% (v/v) glycerol, 0.05% xylene cyanol, 0.05% bromphenol blue, placed on ice, and loaded immediately onto a 5% native polyacrylamide gel (acrylamide-bisacrylamide 37.5:1). Gels were electrophoresed at 11.4 V/cm in 6.7 mm Tris acetate, pH 7.5, 1 mm EDTA, and 32P-labeled protein-DNA complexes visualized by autoradiography after drying. Surface plasmon resonance measurements used a BIAcore 2000. Streptavidin SA sensor chips (Pharmacia Biosensor) were preconditioned according to the manufacturer’s protocol and then derivatized with about 100 response units of a biotinylated 31-bp G-T heteroduplex that has been described previously (11Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar). Human MutSα was bound to the derivatized chip by injecting a 50 nm solution of the heterodimer at a flow rate of 15 μl/min in HBS buffer (10 mm HEPES-KOH, pH 7.4, 0.15 m NaCl, 3.4 mm EDTA, and 0.005% surfactant P20) containing 9 mm MgCl2. Immediately after the association phase was complete, the chip was washed at 15 μl/min with the HBS-MgCl2 buffer, or with this buffer containing ATP, ADP, or AMPPNP as indicated. Measurements were performed at 25 °C, and samples were maintained at 4 °C prior to injection. The heteroduplex-modified SA chip was regenerated by a 20-μl injection of 0.5% sodium dodecyl sulfate. The translocation and molecular switch models postulate different roles for ATP binding and hydrolysis in the function of MutS homologs. In the translocation model, ATP binding and hydrolysis cycles the mismatch recognition activity between several conformational states that are involved in departure of the protein from the mismatch and movement along the helix contour (11Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar). By contrast, the molecular switch model invokes the MutS·ADP complex as the mismatch binding species, with ATP binding subsequent to repair complex assembly promoting release of the protein from the heteroduplex so that excision repair may occur (10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). One potential problem with the molecular switch model is that bacterial, mammalian, and yeast MutS activities bind with high specificity to mismatched base pairs in the absence of exogenous nucleotide (7Drummond J.T. Li G.-M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (538) Google Scholar, 8Iaccarino I. Palombo F. Drummond J. Totty N.F. Hsuan J.J. Modrich P. Jiricny J. Curr. Biol. 1996; 6: 484-486Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 9Alani E. Mol. Cell. Biol. 1996; 16: 5604-5615Crossref PubMed Scopus (142) Google Scholar, 17Su S.-S. Lahue R.S. Au K.G. Modrich P. J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar, 18Su S.-S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (262) Google Scholar, 19Palombo F. Gallinari P. Iaccarino I. Lettieri T. Hughes M. D’Arrigo A. Truong O. Hsuan J.J. Jiricny J. Science. 1995; 268: 1912-1914Crossref PubMed Scopus (481) Google Scholar). Furthermore, ADP has no significant effect on the apparent affinity of human or yeast MutSα for a mispair (10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar,20Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 9837-9841Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). However, it is possible that these MutS activities may purify as the ADP complex. To address this possibility, we have tested near homogeneous hMutSα isolated from HeLa cells for presence of adenine nucleotides. Analysis of several independent hMutSα preparations using a bioluminescent assay (see “Experimental Procedures”) indicated presence of 0.003 mol of ATP and 0.2 mol of ADP per mol of the MSH2·MSH6 heterodimer. The presence of nucleotide in hMutSα preparations was expected since the protein is purified by a two-step procedure, the first of which involves elution from DNA cellulose by ATP and Mg2+ (7Drummond J.T. Li G.-M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (538) Google Scholar). In fact, ATP and ADP present in hMutSα preparations were reduced by more than 90% by gel filtration spin column chromatography (see “Experimental Procedures”). As shown in Fig. 1, nucleotide depletion had no effect on the ability of hMutSα to bind specifically to G-T heteroduplex DNA, as judged by restriction endonuclease protection assay. Therefore, ADP is not required for hMutSα·mismatch binding. In contrast to ATP, which reduces the affinity of hMutSα for a mismatch (7Drummond J.T. Li G.-M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (538) Google Scholar, 8Iaccarino I. Palombo F. Drummond J. Totty N.F. Hsuan J.J. Modrich P. Jiricny J. Curr. Biol. 1996; 6: 484-486Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), ADP does not alter the specific equilibrium affinity of the protein for heteroduplex DNA to a significant degree (10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). However, ADP does alter the dynamics of hMutSα· heteroduplex interaction as judged by several criteria. Fig. 2 (upper panel) shows the dissociation kinetics of specific hMutSα complexes with a 31-bp G-T heteroduplex as visualized by surface plasmon resonance spectroscopy. Dissociation kinetics monitored by this method were biphasic, and results were fit to a sum of two exponentials. Dissociation rate constants corresponding to the major amplitude, which represented 72–97% of the protein·DNA complexes, are summarized in Table I. In the presence of Mg2+ but in the absence of nucleotide, the major species dissociated with a half-life of about 5 min. Inclusion of 1 mm ATP increased the off-rate about 200-fold. This ATP effect is independent of hydrolysis since 1 mm AMPPNP or 1 mm ATP in the absence of Mg+2 enhanced the dissociation rate to a similar degree, results consistent with previous findings suggesting that ATP binding is sufficient for mismatch dissociation in the case of bacterial MutS (11Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar) and hMutSα (5Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (145) Google Scholar, 10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The presence of ADP also resulted in a large increase in the rate of dissociation of hMutSα·heteroduplex complexes: 1 mm ADP decreased the half-life of specific complexes 25-fold relative to that observed with buffer alone (Table I).Table IKinetics of MutSα·heteroduplex dissociation promoted by nucleotide cofactorsNucleotide (1 mm)Amplitudek obst 1/2fractions−1sBuffer only0.930.002 ± 0.00001350 ± 2ATP0.970.42 ± 0.011.7 ± 0.04ATP (−Mg2+)0.880.45 ± 0.011.5 ± 0.03AMPPNP0.720.33 ± 0.012.1 ± 0.06ADP0.860.05 ± 0.00114 ± 0.3k obs and amplitude values were obtained by nonlinear least squares fit (27Marquardt D.W. J. Soc. Indust. Appl. Math. 1963; 11: 431-441Crossref Google Scholar) of the data of Fig. 2. As described in the legend to Fig. 2, dissociation of the hMutSα·G-T heteroduplex was biphasic. The rate constants shown are for dissociation of the major species, which represented 72–97% of the material (Amplitude). Open table in a new tab k obs and amplitude values were obtained by nonlinear least squares fit (27Marquardt D.W. J. Soc. Indust. Appl. Math. 1963; 11: 431-441Crossref Google Scholar) of the data of Fig. 2. As described in the legend to Fig. 2, dissociation of the hMutSα·G-T heteroduplex was biphasic. The rate constants shown are for dissociation of the major species, which represented 72–97% of the material (Amplitude). Surface plasmon resonance analysis demonstrated that the increased rate of hMutSα·heteroduplex dissociation is a saturable function of ATP concentration. As shown in Fig. 2, the rate of complex dissociation increases hyperbolically with ATP concentration, with aK m of 37 μm, a value very similar to the K m for the nucleotide in the DNA-stimulated ATPase reaction (10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 21Blackwell L.J. Bjornson K.P. Modrich P. J. Biol. Chem. 1998; 273: 32049-32054Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). NheI restriction endonuclease protection assay confirmed saturability of ATP-promoted dissociation and also indicated saturability for dissociation promoted by AMPPNP and ADP (Fig. 3). In this assay, preformed hMutSα·heteroduplex complexes were challenged with NheI and nucleotide, and incubation continued for 5 min (see “Experimental Procedures”). Although individual K 1/2 values obtained by this method cannot be interpreted analytically due to the fact that dissociation occurs during the NheI assay period, comparison of K 1/2 values provides an estimate of the relative efficacy of the different nucleotides for promoting dissociation of the hMutSα· heteroduplex complex. The relativeK 1/2 values for ADP and AMPPNP determined in this way are about 5 and 100 times greater than that for ATP, respectively (Fig. 3). The low relative affinity of the hMutSα·heteroduplex complex for AMPPNP is in accord with previous findings, indicating that this nucleotide is less effective than ATP in suppressing the formation of specific complexes with oligonucleotide heteroduplexes (5Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (145) Google Scholar, 7Drummond J.T. Li G.-M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (538) Google Scholar, 10Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). To further clarify the roles of ATP and ADP in the dynamics of hMutSα-DNA interaction, kinetic competition studies were performed. In these experiments, preformed complexes of hMutSα and a 50-bp [32P]G-T heteroduplex were challenged with a 20-fold molar excess of unlabeled 50-bp heteroduplex or homoduplex DNA in the presence of ADP or ATP (Fig. 4). In the absence of nucleotide, only a modest reduction in preformed hMutSα·DNA complexes was observed (compare lanes 2 and 3 with lane 1), consistent with the kinetic" @default.
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• W2067215249 date "1998-11-01" @default.
• W2067215249 modified "2023-10-18" @default.
• W2067215249 title "Nucleotide-promoted Release of hMutSα from Heteroduplex DNA Is Consistent with an ATP-dependent Translocation Mechanism" @default.
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