{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T06:24:28Z","timestamp":1779344668341,"version":"3.51.4"},"reference-count":37,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5298","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1997,1,17]]},"abstract":"<jats:p>\n            The kinetic mechanism by which the DNA repair helicase UvrD of\n            <jats:italic>Escherichia coli<\/jats:italic>\n            unwinds duplex DNA was examined with the use of a series of oligodeoxynucleotides with duplex regions ranging from 10 to 40 base pairs. Single-turnover unwinding experiments showed distinct lag phases that increased with duplex length because partially unwound DNA intermediate states are highly populated during unwinding. Analysis of these kinetics indicates that UvrD unwinds duplex DNA in discrete steps, with an average \u201cstep size\u201d of 4 to 5 base pairs (approximately one-half turn of the DNA helix). This suggests an unwinding mechanism in which alternating subunits of the dimeric helicase interact directly with duplex DNA.\n          <\/jats:p>","DOI":"10.1126\/science.275.5298.377","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:50:55Z","timestamp":1027763455000},"page":"377-380","source":"Crossref","is-referenced-by-count":217,"title":["Kinetic Measurement of the Step Size of DNA Unwinding by\n            <i>Escherichia coli<\/i>\n            UvrD Helicase"],"prefix":"10.1126","volume":"275","author":[{"given":"Janid A.","family":"Ali","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box B8231, St. Louis, MO 63110, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Timothy M.","family":"Lohman","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box B8231, St. Louis, MO 63110, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1146\/annurev.bi.65.070196.001125","volume":"65","author":"Lohman T. M.","year":"1996","unstructured":"Lohman T. M., Bjornson K. P., Annu. Rev. Biochem. 65, 169 (1996).","journal-title":"Annu. Rev. Biochem."},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1002\/bies.950160103","volume":"16","author":"Matson S. W.","year":"1994","unstructured":"Matson S. W., Bean D. W., George J. W., BioEssays 16, 13 (1994).","journal-title":"BioEssays"},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"5616","DOI":"10.1073\/pnas.79.18.5616","volume":"79","author":"Maples V. F.","year":"1982","unstructured":"Maples V. F., Kushner S. R., Proc. Natl. Acad. Sci. U.S.A. 79, 5616 (1982).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.2665076"},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"4925","DOI":"10.1073\/pnas.82.15.4925","volume":"82","author":"Caron P. R.","year":"1985","unstructured":"Caron P. R., Kushner S. R., Grossman L., Proc. Natl. Acad. Sci. U.S.A. 82 4925 (1985);","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_6_3","first-page":"6774.","author":"Husain I.","unstructured":"Husain I. , van Houten B. , Thomas D. C. , Abdel-Monem M. , Sancar A., ibid., p. 6774..","journal-title":"ibid."},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","first-page":"1954","DOI":"10.1126\/science.7801120","volume":"266","author":"Sancar A.","year":"1994","unstructured":"Sancar A., Science 266 1954 (1994);","journal-title":"Science"},{"key":"e_1_3_1_7_3","first-page":"1959","author":"Modrich P.","unstructured":"Modrich P., ibid., p. 1959.","journal-title":"ibid."},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"1579","DOI":"10.1093\/oxfordjournals.jbchem.a135631","volume":"99","author":"Yamamoto Y.","year":"1986","unstructured":"Yamamoto Y., et al., J. Biochem. 99, 1579 (1986).","journal-title":"J. Biochem."},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1021\/bi00053a028","volume":"32","author":"Runyon G. T.","year":"1993","unstructured":"Runyon G. T., Wong I., Lohman T. M., Biochemistry 32, 602 (1993).","journal-title":"Biochemistry"},{"key":"e_1_3_1_10_2","unstructured":"Ali J. Lohman T. M. in preparation."},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1111\/j.1365-2958.1992.tb00831.x","volume":"6","author":"Lohman T. M.","year":"1992","unstructured":"Lohman T. M., Mol. Microbiol. 6 5 1992;","journal-title":"Mol. Microbiol."},{"key":"e_1_3_1_11_3","doi-asserted-by":"crossref","first-page":"2269","DOI":"10.1016\/S0021-9258(18)53765-0","volume":"268","year":"1993","unstructured":"J. Biol. Chem. 268, 2269 (1993).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_12_2","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1006\/jmbi.1996.0585","volume":"263","author":"Bjornson K. P.","year":"1996","unstructured":"Bjornson K. P., Wong I., Lohman T. M., J. Mol. Biol. 263, 411 (1996).","journal-title":"J. Mol. Biol."},{"key":"e_1_3_1_13_2","doi-asserted-by":"crossref","first-page":"10169","DOI":"10.1016\/S0021-9258(18)67506-4","volume":"261","author":"Matson S. W.","year":"1986","unstructured":"Matson S. W., J. Biol. Chem. 261, 10169 (1986).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_14_2","first-page":"17502","volume":"264","author":"Runyon G. T.","year":"1989","unstructured":"Runyon G. T., Lohman T. M., ibid. 264, 17502 (1989);","journal-title":"ibid."},{"key":"e_1_3_1_14_3","doi-asserted-by":"crossref","first-page":"6383","DOI":"10.1073\/pnas.87.16.6383","volume":"87","author":"Runyon G. T.","year":"1990","unstructured":"Runyon G. T. , Bear D. G. , Lohman T. M., Proc. Natl. Acad. Sci. U.S.A. 87, 6383 (1990).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_15_2","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1093\/nar\/15.2.465","volume":"15","author":"Gilchrist C. A.","year":"1987","unstructured":"Gilchrist C. A., Denhardt D. T., Nucleic Acids Res. 15, 465 (1987).","journal-title":"Nucleic Acids Res."},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"1165","DOI":"10.1016\/0022-2836(91)90926-W","volume":"221","author":"Chao K.","year":"1991","unstructured":"Chao K., Lohman T. M., J. Mol. Biol. 221, 1165 (1991).","journal-title":"J. Mol. Biol."},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.256.5055.350"},{"key":"e_1_3_1_18_2","doi-asserted-by":"crossref","first-page":"20386","DOI":"10.1016\/S0021-9258(20)80740-6","volume":"268","author":"Wong I.","year":"1993","unstructured":"Wong I., Amaratunga M., Lohman T. M., J. Biol. Chem. 268, 20386 (1993).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_19_2","unstructured":"UvrD protein was purified and its concentration determined spectrophotometrically (8). Oligodeoxynucleotides were synthesized and purified and dsDNA substrates (5\u2032 end-labeled on the top strand with 32 P) were prepared as described (31). DNA substrates had a (dT) 40 ssDNA attached to the 3\u2032 end of the bottom strand. Oligodeoxythymidylates were used to avoid intramolecular base pairing within the ssDNA. A 40-nucleotide 3\u2032 ssDNA is optimal for the initiation of DNA unwinding by UvrD in vitro and a 5\u2032 ssDNA tail does not facilitate initiation of DNA unwinding (9)."},{"key":"e_1_3_1_20_2","unstructured":"Buffer U is 25 mM tris-HCl pH 7.5 6 mM NaCl 2.5 mM MgCl 2 5 mM 2-mercaptoethanol 10% (v\/v) glycerol and 0.1 mg\/ml bovine serum albumin (BSA). Buffer A is 25 mM tris pH 7.5 at 25\u00b0C 6 mM NaCl 5 mM 2-mercaptoethanol and 10% (v\/v) glycerol."},{"key":"e_1_3_1_21_2","unstructured":"Kinetics experiments were performed (25\u00b0C buffer U) with a three-syringe quenched-flow apparatus (KinTek RQF-3 University Park PA). UvrD at twice the final concentration was premixed with DNA substrate (2 nM) in buffer A plus 2 mM MgCl 2 and BSA (0.2 mg\/ml) incubated on ice for 20 min and then loaded in one loop (45 \u03bcl) of the quench flow. The other loop (45 \u03bcl) contained ATP (twice the final concentration) in buffer A plus 3 mM MgCl 2 and 10 \u03bcM dT(pT) 15 . Samples were incubated for 3 min at 25\u00b0C (incubation times of 6 min did not affect the reaction). Reactions were initiated by rapidly mixing the two solutions yielding buffer U and quenched after times from 2 ms to 100 s by the addition of 0.4 M Na 3 EDTA in 10% (v\/v) glycerol. The fraction of ssDNA at time t = 0 was determined by mixing the UvrD-DNA solution with buffer A plus 3 mM MgCl 2 (without ATP). Quenched samples were analyzed by nondenaturing 20% polyacrylamide gel electrophoresis to separate ds- from ssDNA. Quenching with Na 3 EDTA also caused dissociation of UvrD from the DNA so that the DNA was deproteinated before electrophoresis. Radioactivity in each band was quantitated with a Betascope 603 blot analyzer (Betagen Waltham MA) and the fraction of DNA duplexes unwound at each time was calculated as described (30). The low DNA substrate concentration (1 nM) prevented reannealing of fully unwound DNA. The amplitude and rate of the lag phase were independent of the dT(pT) 15 concentration (0 to 25 \u03bcM)."},{"key":"e_1_3_1_22_2","doi-asserted-by":"crossref","first-page":"14306","DOI":"10.1021\/bi00251a044","volume":"33","author":"Bjornson K. P.","year":"1994","unstructured":"Bjornson K. P., Amaratunga M., Moore K. J. M., Lohman T. M., Biochemistry 33, 14306 (1994).","journal-title":"Biochemistry"},{"key":"e_1_3_1_23_2","unstructured":"Single-turnover unwinding rates (measured with DNA substrates I and II) were independent of the UvrD concentration [3 nM to 150 nM (monomer)]; hence unwinding initiates from prebound complexes. The apparent equilibrium dissociation constant for UvrD binding to the substrate is 1 to 2 nM; hence at 80 nM UvrD all the DNA is bound to UvrD."},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","DOI":"10.1017\/CBO9780511626203","volume-title":"Kinetics for the Life Sciences. Receptors, Transmitters and Catalysts","author":"Gutfreund H.","year":"1995","unstructured":"Gutfreund H., Kinetics for the Life Sciences. Receptors, Transmitters and Catalysts (Cambridge Univ. Press, Cambridge, England, 1995)."},{"key":"e_1_3_1_25_2","unstructured":"This assumption is reasonable because each DNA substrate has a similar base composition (high G+C content) and the time courses with different length duplexes can be globally fitted to Eq. 1 with the same k obs and step size. Also experiments with two 18-bp substrates (II and V in Table 1) differing in A+T content both fit to mechanisms with n = 4 steps with similar k obs . Global analysis of simulated time courses also showed that the step size determination was not influenced when the unwinding rate constants of alternate steps differed by twofold although k obs was affected."},{"key":"e_1_3_1_26_2","unstructured":"Even in the presence of a high concentration of dT(pT) 15 a second slower unwinding phase with a small amplitude remains which represents unwinding by UvrD bound in a nonproductive form that must first isomerize with rate constant k NP to form a productive complex."},{"key":"e_1_3_1_27_2","unstructured":"The first term in Eq. 1 is the fraction of DNA molecules unwound by the productive (U-DNA) L complexes (23) where x = (U-DNA) L \/[(U-DNA) L + (U-DNA) NP )]; thus xA L = A 1 is the amplitude of the lag phase. The second term in Eq. 1 with amplitude (1 \u2212 x ) A L reflects slower unwinding by nonproductive complexes (25)."},{"key":"e_1_3_1_28_2","unstructured":"Nonlinear least squares analyses were performed with Scientist (MicroMath Scientific Software Salt Lake City UT) and plotted with KaleidaGraph (Synergy Software Reading PA). Uncertainties are reported as 95% confidence limits."},{"key":"e_1_3_1_29_2","unstructured":"Because of the second slow phase (25) analysis of a single time course provides only a minimum estimate of the number of steps n. Equally good fits are obtained for greater values of n by decreasing the amplitude and increasing the rate of the lag phase respectively because these changes can be compensated by increases in both the amplitude and the value of k NP for the slow phase. However simultaneous analysis of all four time courses in Fig. 2A (floating each value of A L ) and globally fitting for the same values of x k obs and k NP provides additional constraints on the upper limit of n for each duplex assuming the step size m = L\/n is independent of L. Global fits with m = 2 or 3 gave poorer fits."},{"key":"e_1_3_1_30_2","unstructured":"Analysis of data in Fig. 2B assumed a constant average step size of m = L\/n = 4.4 and that the amplitudes A 1 are a smooth function of duplex length; however A 1 may decrease in a step-wise manner with L and this may contribute to our noninteger (4.4) estimate of the step size."},{"key":"e_1_3_1_31_2","doi-asserted-by":"crossref","first-page":"6815","DOI":"10.1021\/bi00078a003","volume":"32","author":"Amaratunga M.","year":"1993","unstructured":"Amaratunga M., Lohman T. M., Biochemistry 32, 6815 (1993).","journal-title":"Biochemistry"},{"key":"e_1_3_1_32_2","doi-asserted-by":"crossref","first-page":"7596","DOI":"10.1016\/S0021-9258(18)42558-6","volume":"267","author":"Wong I.","year":"1992","unstructured":"Wong I., Chao K. L., Bujalowski W., Lohman T. M., J. Biol. Chem. 267, 7596 (1992).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_33_2","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/0003-2697(83)90660-7","volume":"130","author":"Barshop B. A.","year":"1983","unstructured":"Barshop B. A., Wrenn R. F., Frieden C., Anal. Biochem. 130, 134 (1983).","journal-title":"Anal. Biochem."},{"key":"e_1_3_1_34_2","unstructured":"We thank I. Wong K. Bjornson and K. Moore for critical discussions; M. Amaratunga for preliminary experiments; W. van Zante and T. Ho for DNA synthesis; and P. Burgers R. Gregorian and J. Hsieh for comments on the manuscript. Supported in part by NIH grant GM45948."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.275.5298.377","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T04:58:29Z","timestamp":1705121909000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.275.5298.377"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1997,1,17]]},"references-count":37,"journal-issue":{"issue":"5298","published-print":{"date-parts":[[1997,1,17]]}},"alternative-id":["10.1126\/science.275.5298.377"],"URL":"https:\/\/doi.org\/10.1126\/science.275.5298.377","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1997,1,17]]}}}