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• W2020143772 abstract "Bloom's syndrome is a rare autosomal recessive disorder characterized by genomic instability and predisposition to cancer. BLM, the gene defective in Bloom's syndrome, encodes a 159-kDa protein possessing DNA-stimulated ATPase and ATP-dependent DNA helicase activities. We have examined mechanistic aspects of the catalytic functions of purified recombinant BLM protein. Through analyzing the effects of different lengths of DNA cofactor on ATPase activity, we provide evidence to suggest that BLM translocates along single-stranded DNA in a processive manner. The helicase reaction catalyzed by BLM protein was examined as a function of duplex DNA length. We show that BLM catalyzes unwinding of short DNA duplexes (≤71 base pairs (bp)) but is severely compromised on longer DNA duplexes (≥259-bp). The presence of the human single-stranded DNA-binding protein (human replication protein A (hRPA)) stimulates the BLM unwinding reaction on the 259-bp partial duplex DNA substrate. Heterologous single-stranded DNA-binding proteins fail to stimulate similarly the helicase activity of BLM protein. This is the first demonstration of a functional interaction between BLM and another protein. Consistent with a functional interaction between hRPA and the BLM helicase, we demonstrate a direct physical interaction between the two proteins mediated by the 70-kDa subunit of RPA. The interactions between BLM and hRPA suggest that the two proteins function togetherin vivo to unwind DNA duplexes during replication, recombination, or repair. Bloom's syndrome is a rare autosomal recessive disorder characterized by genomic instability and predisposition to cancer. BLM, the gene defective in Bloom's syndrome, encodes a 159-kDa protein possessing DNA-stimulated ATPase and ATP-dependent DNA helicase activities. We have examined mechanistic aspects of the catalytic functions of purified recombinant BLM protein. Through analyzing the effects of different lengths of DNA cofactor on ATPase activity, we provide evidence to suggest that BLM translocates along single-stranded DNA in a processive manner. The helicase reaction catalyzed by BLM protein was examined as a function of duplex DNA length. We show that BLM catalyzes unwinding of short DNA duplexes (≤71 base pairs (bp)) but is severely compromised on longer DNA duplexes (≥259-bp). The presence of the human single-stranded DNA-binding protein (human replication protein A (hRPA)) stimulates the BLM unwinding reaction on the 259-bp partial duplex DNA substrate. Heterologous single-stranded DNA-binding proteins fail to stimulate similarly the helicase activity of BLM protein. This is the first demonstration of a functional interaction between BLM and another protein. Consistent with a functional interaction between hRPA and the BLM helicase, we demonstrate a direct physical interaction between the two proteins mediated by the 70-kDa subunit of RPA. The interactions between BLM and hRPA suggest that the two proteins function togetherin vivo to unwind DNA duplexes during replication, recombination, or repair. Bloom's syndrome base pair replication protein A human RPA single-stranded binding protein E. coli SSB ssDNA, single-stranded DNA bovine serum albumin phosphate-buffered saline enzyme-linked immunosorbent assay nucleotide Bloom's syndrome (BS)1is a rare autosomal recessive disorder characterized by pre- and postnatal growth retardation, immunodeficiency, sun-induced facial erythema, and a greatly increased predisposition to a wide range of malignant cancers (1German J. Medicine (Baltimore). 1993; 72: 393-406Crossref PubMed Scopus (451) Google Scholar). The most characteristic feature of cells from BS patients is genomic instability (for review, see Ref. 2Chakraverty R.K. Hickson I.D. BioEssays. 1999; 21: 286-294Crossref PubMed Scopus (196) Google Scholar). This is manifested predominantly as an elevated frequency of chromosome breaks and exchanges (1German J. Medicine (Baltimore). 1993; 72: 393-406Crossref PubMed Scopus (451) Google Scholar, 3Ellis N.A. German J. Hum. Mol. Genet. 1996; 5: 1457-1463Crossref PubMed Google Scholar, 4Watt P.M. Hickson I.D. Borts R.H. Louis E.J. Genetics. 1996; 144: 935-945Crossref PubMed Google Scholar) as well as a characteristic increase in the level of reciprocal exchanges between sister chromatids (5Langlois R.G. Bigbee W.L. Jensen R.H. German J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 670-674Crossref PubMed Scopus (180) Google Scholar). BS cells exhibit hyper-recombination (1German J. Medicine (Baltimore). 1993; 72: 393-406Crossref PubMed Scopus (451) Google Scholar, 6Cheng R.Z. Murano S. Kurz B. Shmookler R.R. Mutat. Res. 1990; 237: 259-269Crossref PubMed Scopus (59) Google Scholar, 7Ellis N.A. Groden J. Ye T.Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1200) Google Scholar) and abnormalities in DNA replication that include an extended S phase and accumulation of abnormal replication intermediates compared with normal cells (8Gianneli F. Benson P.F. Pawsey S.A. Polani P.E. Nature. 1977; 265: 466-469Crossref PubMed Scopus (131) Google Scholar, 9Hanaoka F. Yamada M. Takeuchi F. Goto M. Miyamoto T. Hori T. Adv. Exp. Med. Biol. 1985; 190: 439-457Crossref PubMed Scopus (46) Google Scholar, 10Lonn U. Lonn S. Nylen U. Winblad G. German J. Cancer Res. 1990; 50: 3141-3145PubMed Google Scholar). The gene defective in BS, designated BLM, encodes a protein of 1417 amino acids with the seven conserved motifs found in RNA and DNA helicases (7Ellis N.A. Groden J. Ye T.Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1200) Google Scholar). By sequence alignment, the BLM gene product belongs to the RecQ subfamily of DNA helicases that includes a singleEscherichia coli DNA helicase named RecQ, a protein required for the RecF pathway of genetic recombination (11Nakayama K. Irino N. Nakayama H. Mol. Gen. Genet. 1985; 200: 266-271Crossref PubMed Scopus (123) Google Scholar) and for suppression of illegitimate recombination (12Hanada K. Ukita T. Kohno Y. Saito K. Kato J. Ikeda H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3860-3865Crossref PubMed Scopus (239) Google Scholar). Two yeast proteins,Saccharomyces cerevisiae Sgs1p (13Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (615) Google Scholar, 14Watt P.M. Louis E.J. Borts R.H. Hickson I.D. Cell. 1995; 81: 253-260Abstract Full Text PDF PubMed Scopus (376) Google Scholar) and Schizosaccharomyces pombe Rqh1p (15Stewart E. Chapman C.R. Al-Khodairy F. Carr A.M. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (326) Google Scholar), belong to the RecQ subfamily and have proposed roles in recombination and possibly replication. At present, five human members of the RecQ subfamily have been identified, including BLM (7Ellis N.A. Groden J. Ye T.Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1200) Google Scholar), WRN (16Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1473) Google Scholar), RecQL (17Seki M. Miyazawa H. Tada S. Yanagisawa J. Yamaoka T. Hoshino S. Ozawa K. Eki T. Nogami M. Okumura K. Nucleic Acids Res. 1994; 22: 4566-4573Crossref PubMed Scopus (143) Google Scholar), RecQL4 (18Kitao S. Ohsugi I. Ichikawa K. Goto M. Furuichi Y. Shimamoto A. Genomics. 1998; 54: 443-452Crossref PubMed Scopus (233) Google Scholar), and RecQL5 (18Kitao S. Ohsugi I. Ichikawa K. Goto M. Furuichi Y. Shimamoto A. Genomics. 1998; 54: 443-452Crossref PubMed Scopus (233) Google Scholar). Mutations in the WRN gene are responsible for the premature aging disorder Werner's syndrome (16Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1473) Google Scholar). Most recently, it was demonstrated that mutations in the RecQL4 gene result in some cases of Rothmund-Thomson's syndrome (19Kitao S. Shimamoto A. Goto M. Miller R.W. Smithson W.A. Lindor N.M. Furuichi Y. Nat. Genet. 1999; 22: 82-84Crossref PubMed Scopus (565) Google Scholar). Both Werner's syndrome (20Salk D. Bryant E. Hoehn H. Johnston P. Martin G.M. Adv. Exp. Med. Biol. 1985; 190: 305-311Crossref PubMed Scopus (62) Google Scholar, 21Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (210) Google Scholar) and Rothmund-Thomson's syndrome (22Lindor N.M. Devries E.M. Michels V.V. Schad C.R. Jalal S.M. Donovan K.M. Smithson W.A. Kvols L.K. Thibodeau S.N. Dewald G.W. Clin. Genet. 1996; 49: 124-129Crossref PubMed Scopus (74) Google Scholar), like BS, are characterized by chromosomal instability suggesting that DNA helicases are important caretakers of the human genome with specialized roles in pathways of DNA metabolism. Biochemical studies have shown that the BLM protein is a DNA-stimulated ATPase and ATP-dependent helicase, catalyzing strand displacement of short and medium length oligonucleotides (≤91 bp) from partial duplex substrates with a 3′ to 5′ polarity (23Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). BLM helicase preferentially unwinds a G4 DNA substrate consisting of four guanine-rich strands stabilized by Hoogsteen bonding (24Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Electron microscopy analysis has shown that BLM protein forms oligomeric rings in solution (25Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Size exclusion chromatography data indicate that the majority of enzymatically active BLM has an apparent molecular mass of >700 kDa, which is consistent with an oligomeric structure for BLM (25Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Aside from these structural and biochemical data, molecular details of the interactions of BLM protein with other proteins and biological DNA substrates remain to be defined. The molecular deficiencies involved in the clinical phenotype of BS presumably reflect an impaired function of the BLM protein in a pathway of nucleic acid metabolism. Transfection of the wild-type BLM gene into BS cells reduces the frequency of sister chromatid exchanges (26Neff N.F. Ellis N.A. Ye T.Z. Noonan J. Huang K. Sanz M. Proytcheva M. Mol. Biol. Cell. 1999; 10: 665-676Crossref PubMed Scopus (124) Google Scholar). Mutant alleles of BLM found in individuals with clinical BS encode BLM protein that is devoid of DNA helicase activity and fails to reduce the high sister chromatid exchanges in transfected BS cells (26Neff N.F. Ellis N.A. Ye T.Z. Noonan J. Huang K. Sanz M. Proytcheva M. Mol. Biol. Cell. 1999; 10: 665-676Crossref PubMed Scopus (124) Google Scholar, 27Bahr A. De Graeve F. Kedinger C. Chatton B. Oncogene. 1998; 17: 2565-2571Crossref PubMed Scopus (54) Google Scholar). These studies provide evidence that the enzymatic activity of BLM is important for its cellular function. In an effort to better understand the mechanistic aspects of the BLM catalytic activities, we have further characterized the catalytic activities of the BLM protein. Our results show that BLM unwinds short DNA duplexes (≤71 bp) but is severely compromised on DNA duplexes ≥259 bp. The poor unwinding of BLM helicase on relatively long DNA duplex substrates suggested to us that additional protein factor(s) might convert the helicase into a more processive enzyme. A good candidate to serve as an accessory factor to BLM helicase is the heterotrimeric single-stranded DNA-binding protein RPA that has been implicated in replication, recombination, and DNA repair (28Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1161) Google Scholar). Evidence indicates that RPA modulates these processes by specific protein-protein and protein-DNA interactions. We have recently demonstrated that a specific functional and physical interaction exists between human RPA and WRN helicase (29Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). WRN helicase was found to be capable of unwinding long DNA duplexes up to 849 bp in a reaction dependent on hRPA. The notion that RPA may coordinately function with BLM helicase in vivo is supported by the recent finding that BLM colocalizes with RPA in meiotic prophase nuclei of mammalian spermatocytes (30Walpita D. Plug A.W. Neff N.F. German J. Ashley T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5622-5627Crossref PubMed Scopus (82) Google Scholar). Colocalization of BLM and RPA at the synaptonemal complex of homologously synapsed autosomal bivalents suggests that these foci mark sites of ssDNA synaptic-related meiotic activity. The interactive roles of BLM and hRPA on synapsed meiotic bivalents are undefined but likely to involve the catalytic activity of BLM protein. In this study we have shown that RPA is required to support BLM helicase activity on a relatively long DNA duplex of 259-bp. Two heterologous SSBs, ESSB and gp32, failed to substitute for RPA. This functional interaction was further substantiated by the demonstration of a physical interaction between hRPA and BLM. This interaction, and colocalization of the two proteins in meiotic cells, suggests that BLM and RPA function together in a pathway of DNA metabolism such as recombination or replication. Recombinant hexahistidine-tagged BLM protein was overexpressed in Saccharomyces cerevisiae and purified as described previously (25Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). hRPA containing all three subunits (RPA70, RPA32, and RPA14) was purified as described previously (31Kenny M.K. Schlegel U. Furneaux H. Hurwitz J. J. Biol. Chem. 1990; 265: 7693-7700Abstract Full Text PDF PubMed Google Scholar). S. cerevisiae replication protein A (scRPA) was a generous gift of Drs. Dan Bean and Steven Matson, University of North Carolina, Chapel Hill. ESSB was purchased from Promega. T4 gp32 was from U. S. Biochemical Corp. Restriction endonuclease HaeIII was obtained from New England Biolabs. Klenow enzyme was obtained from Roche Molecular Biochemicals. DNase I was from Roche Molecular Biochemicals. BSA type V was from ICN Biochemicals. M13mp18 ssDNA was from New England Biolabs. The 28-mer oligonucleotide 5′-TCCCAGTCACGACGTTGTAAAACGACGG-3′ was from Life Technologies, Inc. M13mp18 RFI was prepared as described previously (32Lechner R.L. Richardson C.C. J. Biol. Chem. 1983; 258: 11185-11196Abstract Full Text PDF PubMed Google Scholar). (dT)∼263 and ATP were from Amersham Pharmacia Biotech. (dT)∼900 was from Midland Certified Reagent Co. [3H]ATP was from Amersham Pharmacia Biotech, and [α-32P]dCTP was from NEN Life Science Products. The 71-, 259-, and 851-bp M13mp18 partial duplex substrates were constructed as described previously (29Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar,33Matson S.W. George J.W. J. Biol. Chem. 1987; 262: 2066-2076Abstract Full Text PDF PubMed Google Scholar) with the following modifications. Duplex DNA fragments (69-, 257-, and 849-bp) from the HaeIII digest of M13mp18 replicative form were purified by polyacrylamide gel electrophoresis and electroelution. The desired restriction fragment (100 ng) and M13mp18 ssDNA circle (2 μg) were incubated together in an annealing reaction. The resulting partial duplex was labeled at its 3′ terminus in a fill-in reaction with [32P]dCTP and Klenow enzyme. The 30-bp M13mp18 partial duplex substrate was constructed with a 28-mer complementary to positions 6296–6323 in M13mp18. The 28-mer was annealed to M13mp18 ssDNA circle and labeled at its 3′ end as described above. Partial duplex DNA substrates were purified by gel filtration column chromatography using Bio-Gel A-5M resin (Bio-Rad). Helicase assay reaction mixtures (20 μl) contained 50 mm Tris-HCl (pH 7.4), 5 mmMgCl2, 5 mm ATP, 100 μg/ml bovine serum albumin, 50 mm NaCl, and the indicated amounts of BLM helicase and/or single-stranded DNA-binding protein. The concentration of the 30-, 71-, 259-, and 851-bp partial duplex helicase substrates in the reaction mixture was approximately 2 μm (nucleotide). Reactions were initiated by the addition of BLM protein and incubated at 37 °C for the indicated times. Reactions were terminated by the addition of 10 μl of 50 mm EDTA, 40% glycerol, 0.9% SDS, 0.1% bromphenol blue, 0.1% xylene cyanol. The products of helicase reactions with the 30-, 71-, 259-, and 851-bp partial duplex substrates were resolved on 12, 8, 6, and 6% nondenaturing polyacrylamide gels, respectively, as described previously (29Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Radiolabeled DNA species in polyacrylamide gels were visualized using a PhosphorImager or film autoradiography and quantitated using the ImageQuant software (Molecular Dynamics). The percent helicase substrate unwound was calculated by the following formula: % displacement = 100 × P/(S + P). P is the product volume and S is the substrate volume. The values for P and S have been corrected after subtracting background values in the no enzyme and heat-denatured controls, respectively. All helicase data represent the average of at least three independent determinations. Standard ATPase assay reaction mixtures (30 μl) contained 50 mm Tris-HCl (pH 7.4), 5 mmMgCl2, the indicated ssDNA effector (30 μmnucleotide), 0.8 mm [3H]ATP (42 cpm/pmol), 13 nm BLM protein, and the indicated amounts of hRPA. Reactions were initiated by the addition of BLM protein and incubated at 37 °C. Samples (5 μl) were removed at 2-min intervals and evaluated by thin layer chromatography as described previously (34Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar). Less than 20% of the substrate ATP was consumed in the reaction over the entire time course of the experiment. The kinetic rate constant (k cat) values were expressed as the mean of at least three independent determinations. hRPA was diluted to a concentration of 1.65 ng/μl in Carbonate Buffer (0.016 mNa2CO3, 0.034 m NaHCO3(pH 9.6)). hRPA was then added to the appropriate wells of a 96-well ELISA plate (100 μl/well) and allowed to incubate for 2 h at 24 °C. For control experiments, BSA was substituted for hRPA in the coating step. Wells were aspirated and washed three times with Wash Buffer (PBS, 0.5% Tween 20). Blocking Buffer (PBS, 0.5% Tween 20, 3% BSA) was added to appropriate wells and allowed to incubate 2 h at 24 °C. Wells were aspirated and washed one time with Blocking Buffer. BLM protein was diluted to 1.0 ng/μl in Binding Buffer (50 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 5 mm ATP, 100 μg/ml BSA, and 50 mm NaCl, or the indicated NaCl concentration). The diluted BLM protein was then added to appropriate wells of the ELISA plate (100 μl/well) and allowed to incubate for 30 min at 24 °C. Wells were aspirated and washed three times with Binding Buffer. Primary antibody (rabbit polyclonal IgG against BLM protein) was diluted 1:1000 in Blocking Buffer, added to appropriate wells, and allowed to incubate 1 h at 24 °C. Wells were aspirated and washed four times with Blocking Buffer. Secondary antibody (goat anti-rabbit IgG-horseradish peroxidase) (Jackson ImmunoResearch) was diluted 1:10,000 in Conjugate Buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 0.05% Tween 20, 1% BSA), added to appropriate wells, and allowed to incubate 30 min at 24 °C. Wells were aspirated and washed five times with Conjugate Buffer. Complexes were detected using K-Blue substrate (Biogen Corp.). The reaction was terminated after 1 min with 1n sulfuric acid. Absorbance readings were taken at 450 nm. The A 450 values, corrected for background signal in the presence of BSA, are expressed as the mean of three independent determinations. For DNase I treatment, both BLM protein and hRPA were pretreated with 20 units of DNase I in Binding Buffer at 37 °C for 15 min. The proteins were subsequently used in the ELISA as described above. Under the conditions used for DNase I treatment, 250 ng of control DNA standard (DNA Molecular Weight Marker II, Roche Molecular Biochemicals) was completely degraded (<2.5 ng, detectable limit) as evidenced by SYBR Green Stain (FMC Bioproducts) detection of DNA electrophoresed on a 1% agarose gel. The fraction of the immobilized hRPA bound to the microtiter well that was specifically bound by BLM protein was determined from the ELISAs. A Hill plot was used to analyze the data (Equations 1 and 2).Kd=(1−f)[Pt]fEquation 1 log[Pt]=log(f/(1−f))+logKdEquation 2 Kd is the dissociation constant of the BLM·hRPA complex, [Pt] is the total concentration of BLM protein present in the reaction, and f is the ratio of the amount of the bound hRPA over the total amount of hRPA in the reaction. The logarithm of [Pt] was plotted against the logarithm of (f/(1 − f)), and the y intercept represented the logarithm of Kd. Far Western blotting analysis was conducted essentially as described by Wu et al. (35Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.-F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9646Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Previously, a physical interaction between BLM and hTOPIIIα was demonstrated (35Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.-F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9646Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). In these studies, the BLM-hTOPIIIα interaction served as a positive control in experiments to detect a BLM-hRPA interaction. Briefly, 0.2–1.0 μg of each polypeptide was subjected to SDS-polyacrylamide gel electrophoresis and transferred to Hybond-ECL filters (Amersham Pharmacia Biotech). All subsequent steps were performed at 4 °C. Filters were immersed twice in denaturation buffer (6 m guanidine HCl in PBSA) for 10 min followed by 6 times for 10 min in serial dilutions (1:1) of denaturation buffer supplemented with 1 mm dithiothreitol. Filters were blocked in TBS containing 10% powdered milk, 0.3% Tween 20 for 30 min before being incubated in BLM (0.5 μg/ml) in TBS supplemented with 0.25% powdered milk, 0.3% Tween 20, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride for 60 min. Filters were washed for 4 times for 10 min in TBS containing 0.3% Tween 20, 0.25% powdered milk. The second wash contained 0.0001% glutaraldehyde. Conventional Western analysis was then performed to detect the presence of BLM using BFL-103 (35Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.-F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9646Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) as primary antibody. Anti-mouse IgG/horseradish peroxidase conjugate (Sigma) was used as secondary antibody at a 1:10,000 dilution and detected using ECL (Amersham Pharmacia Biotech) following the manufacturer's instructions. BLM helicase, like all helicases characterized to date, hydrolyzes nucleoside triphosphate as an energy source for the unwinding reaction (36Matson S.W. Bean D.W. George J.W. BioEssays. 1994; 16: 13-22Crossref PubMed Scopus (266) Google Scholar). We studied the DNA-stimulated ATPase activity of BLM protein in the presence of DNA cofactors of varying lengths. Results of these assays are summarized in TableI. In the absence of a DNA effector, little or no ATP hydrolysis by BLM protein could be detected using a thin layer chromatography method to measure conversion of [3H]ATP to [3H]ADP. These results are consistent with the strong stimulation of ATP hydrolysis by DNA effectors reported by Karow et al. (23Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). With the very short (dT)16 oligonucleotide, the turnover rate constantk cat for ATP hydrolysis was 141 min−1. As the length of the dT tract was increased to an average length of 263 nt, the k cat increased nearly 6-fold. Increasing the dT tract from 263 to 900 nt resulted in only a modest 1.2-fold increase in the k cat for ATP hydrolysis. By using M13 ssDNA circles as an infinitely long DNA effector, the increase in k cat to 1163 min−1 was similarly modest (1.2-fold). However, we cannot rule out that very long ssDNA molecules would result in a significantly greater ATPase activity of BLM since the M13 ssDNA circle contains secondary structure. These data indicate that the stimulatory effect of DNA molecules on BLM ATP hydrolysis begins to plateau at a poly(dT) tract length of approximately 263 nt.Table IHydrolysis of ATP (kcat) catalyzed by BLM in the presence of various DNA effectorsDNA effector1-aDNA effector concentration was 30 μm nucleotide phosphate unless specified otherwise.kcatmin−1NoneND1-bND, not detectable.(dT)16141 ± 27(dT)∼263822 ± 216(dT)∼900956 ± 57M13mp18 ssDNA circle1163 ± 358(dT)∼900 + dT161-cConcentration of (dT)16 was 90 μm nucleotide phosphate.921 ± 82M13mp18 ssDNA + (dT)161-cConcentration of (dT)16 was 90 μm nucleotide phosphate.1022 ± 183The BLM concentration was 13 nm (monomer).1-a DNA effector concentration was 30 μm nucleotide phosphate unless specified otherwise.1-b ND, not detectable.1-c Concentration of (dT)16 was 90 μm nucleotide phosphate. Open table in a new tab The BLM concentration was 13 nm (monomer). The stimulation of BLM protein ATPase activity by a given DNA concentration (30 μm nucleotide phosphate) was much greater for long dT tracts (263 and 900). To investigate if the BLM ATPase reaction was saturated with respect to each DNA effector, we examined BLM protein ATP hydrolysis in the presence of a 3-fold greater concentration of nucleotide phosphate (90 μm) for each DNA effector. The initial rates of BLM ATP hydrolysis were not increased with the elevated nucleotide phosphate concentration for each of the DNA effectors (data not shown). These data provide evidence that the ATPase reaction is saturated with respect to ssDNA for each DNA effector under the reaction conditions used. The data in Table I might be interpreted to suggest that the free ends of the DNA effector molecules are inhibitory to the ATP hydrolysis reaction of BLM protein. To address this possibility, we mixed (dT)16 with either (dT)∼900 or M13 ssDNA at a 3:1 molar ratio of nucleotide phosphate and tested the mixture in BLM ATPase reactions. The k cat values for BLM-catalyzed ATP hydrolysis in the presence of the (dT)∼900 + (dT)16 mixture or M13 ssDNA + (dT)16 mixture were 921 and 1022 min−1, respectively (Table I). These data indicate that the BLM ATPase reaction stimulated by (dT)∼900 or M13 ssDNA was not inhibited by the presence of the short (dT)16 molecules, suggesting that the DNA ends do not inhibit ATP hydrolysis. Helicases can be classified by the macroscopic reaction mechanism for unwinding (36Matson S.W. Bean D.W. George J.W. BioEssays. 1994; 16: 13-22Crossref PubMed Scopus (266) Google Scholar). Duplex unwinding as a function of duplex length is an important property of each helicase and may yield insights into the biochemical role of the enzyme in the cell. By using a variety of DNA substrates, biochemical studies of purified helicases in vitro have demonstrated that each DNA unwinding enzyme exhibits its own characteristic dependence of unwinding on DNA duplex length. To characterize the effect of duplex length on the unwinding activity of BLM helicase, we tested partial duplex substrates of varying length in a strand displacement assay. Unwinding of M13 partial duplex DNA substrate molecules of 30, 71, 259, and 851 bp was measured as a function of BLM protein concentration (Fig.1). The figures for percent unwinding of the 30- (Fig. 1 A) and 71-bp (Fig. 1 B) substrates rose to 70 and 37% with 4 nm BLM and 92 and 65% with 16 nm BLM (Fig. 1 C). Further increase in amount of BLM helicase in the reaction did not result in an increase in the percent of DNA unwound for either substrate. The molecular explanation for the inability to achieve a greater percentage of the 71-bp partial duplex DNA substrates unwound is not clear. This phenomenon has been previously observed for UvrD helicase (33Matson S.W. George J.W. J. Biol. Chem. 1987; 262: 2066-2076Abstract Full Text PDF PubMed Google Scholar) and may reflect strand reannealing during the unwinding reaction (discussed below). These results demonstrate that BLM helicase is not a very processive helicase even on short DNA duplexes, since the fraction of partial duplex molecules unwound exhibits a strict dependence on length of duplex. Rather, the amount of BLM helicase that is required in the unwinding reaction for the 30- and 71-bp partial duplex substrates is proportional to the length of DNA duplex to be unwound. It is useful to express the strand displacement data as a rate of base pairs unwound per min per BLM helicase monomer (bp/min/BLM monomer). At a BLM concentration of 2 nm, the rates of" @default.
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