Anhydrous sol-gel condensation of triethyl phosphate [(CH 3 CH2 O) 3 PO] with boron trichloride (BCL 3 ) or triethyl aluminum [(CH 3 CH 2 ) 3 A1]in organic solvents, led to formation of metallophosphate gels. The pore fluid of the gels was removed under supercritical conditions in a pressurized vessel to form aerogels. The aerogels were then calcined at progressively higher temperatures to produce high surface area phosphates. Since the initial gel reagent mixtures contained several NMR active nuclei, the condensation chemistry prior to the gel point was monitored by solution n B NMR. The surface areas, distribution of pore sizes, and total pore volumes of the aerogel products were determined using nitrogen gas physisorption methods.
The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species. The specific activity for ssDNA stimulated ATPase activity was independent of Dda concentration. Dda was mutated to produce an ATPase-deficient protein (K38A Dda) by altering a residue within a conserved, nucleotide binding loop. The helicase activity of K38A Dda was inactivated, although DNA binding properties were similar to Dda. In the presence of limiting DNA substrate, the rate of unwinding by Dda was not changed; however, the amplitude of product formation was reduced in the presence of increasing concentrations of K38A Dda. The reduction was between that expected for a monomeric or dimeric helicase based on simple competition for substrate binding. When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no reduction in the observed rate for strand separation. Taken together, these results indicate that oligomerization of Dda is not required for DNA unwinding. The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species. The specific activity for ssDNA stimulated ATPase activity was independent of Dda concentration. Dda was mutated to produce an ATPase-deficient protein (K38A Dda) by altering a residue within a conserved, nucleotide binding loop. The helicase activity of K38A Dda was inactivated, although DNA binding properties were similar to Dda. In the presence of limiting DNA substrate, the rate of unwinding by Dda was not changed; however, the amplitude of product formation was reduced in the presence of increasing concentrations of K38A Dda. The reduction was between that expected for a monomeric or dimeric helicase based on simple competition for substrate binding. When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no reduction in the observed rate for strand separation. Taken together, these results indicate that oligomerization of Dda is not required for DNA unwinding. double-stranded DNA single-stranded DNA denatured salmon testes DNA dithiobis(succinimidyl propionate) adenosine 5′-O-(3-thiotriphosphate) nucleotide(s) wild type polyacrylamide gel electrophoresis β -mercaptoethanol When dsDNA1 is replicated, repaired, or recombined, the necessary ssDNA intermediates are provided by the activity of helicases (1Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (459) Google Scholar, 2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 3West S.C. Cell. 1996; 86: 177-180Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 4Egelman E. Curr. Biol. 1996; 4: 759-762Google Scholar, 5Matson S.W. Bean D.W. George J.W. Bioessays. 1994; 16: 13-22Crossref PubMed Scopus (269) Google Scholar). These enzymes appear to be ubiquitous, having been identified in viral, bacterial, and eukaryotic systems. Helicases hydrolyze nucleotide triphosphates, usually ATP, to obtain the energy needed to unwind dsDNA. They translocate on DNA often in a very processive manner, unwinding thousands of base pairs in a single binding event. Their processive nature implies that helicases have multiple DNA binding sites; indeed, a characteristic feature of many helicases is their propensity to form oligomeric structures, often dimers or hexamers.Knowledge of the oligomeric form of a helicase is of fundamental concern in development of a mechanism for unwinding activity, and many approaches have been applied to determine oligomeric structure. The lack of evidence for oligomerization from biophysical experiments has led to the suggestion that some helicases function as monomers. PcrA helicase of Bacillus stearothermophilus is proposed to function as a monomer that contains two binding sites for DNA (6Bird L.E. Brannigan J.A. Subramanya H.S. Wigley D.B. Nucleic Acids Res. 1998; 26: 2686-2693Crossref PubMed Scopus (91) Google Scholar, 7Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). Evidence has been provided that suggests Escherichia coli UvrD helicase (helicase II) can be active as a monomerin vivo and in vitro (8Mechanic L.E. Hall M.C. Matson S.W. J. Biol. Chem. 1999; 274: 12488-12498Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The E. coliRep helicase has been proposed to function as a dimer (2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar), whereas other helicases such as T7 gene 4 helicase and T4 gp41 helicase appear to function as hexamers (9Patel S.S. Hingorani M.M. J. Biol. Chem. 1993; 268: 10668-10675Abstract Full Text PDF PubMed Google Scholar, 10Dong F. Gogol E.P. von Hippel P. J. Biol. Chem. 1995; 270: 7462-7473Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).Helicases have been classified according to sequence homology (11Gorbalenya A. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1024) Google Scholar), and those enzymes in superfamily 1 and superfamily 2 have proven difficult to characterize in terms of oligomerization. The focus of this study is the bacteriophage T4 helicase, Dda, a 5′-to-3′ helicase classified in superfamily 1. Evidence suggests that Dda is involved in T4 replication initiation (12Gauss P. Park K. Spencer T.E. Hacker K.J. J. Bacteriol. 1994; 176: 1667-1672Crossref PubMed Google Scholar). T4 Dda− mutants show a delayed DNA synthesis phenotype, consistent with this hypothesis. Dda also enhances the rate of branch migration owing to a specific interaction with the T4 recombinase (UvsX) and is therefore likely to play a role in recombination (13Kodadek T. Alberts B.M. Nature. 1987; 326: 312-314Crossref PubMed Scopus (52) Google Scholar, 14Hacker K.J. Alberts B.M. J. Biol. Chem. 1992; 267: 20674-20681Abstract Full Text PDF PubMed Google Scholar, 15Formosa T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (90) Google Scholar). Dda binds tightly to the T4 single-stranded DNA-binding protein, gp32, although the significance of this interaction has not been determined (15Formosa T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (90) Google Scholar, 16Jongeneel C.V. Bedinger P. Alberts B.M. J. Biol. Chem. 1984; 259: 12933-12938Abstract Full Text PDF PubMed Google Scholar).Dda translocates on ssDNA with a strong directional bias in the 5′-to-3′ direction (17Raney K.D. Benkovic S.J. J. Biol. Chem. 1995; 270: 22236-22242Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It is capable of removing protein blocks placed in the path of the enzyme, including streptavidin bound to biotin-labeled oligonucleotides (18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar). Dda is not highly processive (19Jongeneel C.V. Formosa T. Alberts B.M. J. Biol. Chem. 1984; 259: 12925-12932Abstract Full Text PDF PubMed Google Scholar,20Raney K.D. Sowers L.D. Millar D.P. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6644-6648Crossref PubMed Scopus (127) Google Scholar). Unwinding of only a few hundred base pairs can be prevented by addition of ssDNA, even when Dda is incubated with the substrate prior to addition of the competitor DNA (19Jongeneel C.V. Formosa T. Alberts B.M. J. Biol. Chem. 1984; 259: 12925-12932Abstract Full Text PDF PubMed Google Scholar). In contrast, the replicative helicase from bacteriophage T4, gp41, can unwind thousands of base pairs in a single binding event (21Dong F. Weitzel S.E. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14456-14461Crossref PubMed Scopus (44) Google Scholar). Like T7 gene 4 helicase, gp41 is a hexameric helicase that encircles ssDNA, resulting in very high processivity (18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar, 22Egelman E.H., Yu, X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (251) Google Scholar). The oligomeric nature of Dda was investigated in order to determine whether its relatively low processivity might be related to the oligomeric structure, and to lay the foundation for future mechanistic studies.DISCUSSIONThe search for a functional oligomeric structure of Dda has proven elusive. Experimental approaches such as chemical cross-linking and gel filtration did not strongly support nor eliminate formation of oligomeric species (Figs. 1 and 2). Biochemical assays in which the ATPase activity of Dda was measured at varying enzyme concentration did not provide any evidence for oligomerization (Fig. 3). Hence, methods that have provided support for oligomerization of other helicases gave negative results with Dda. Transient, protein-protein interactions that might be required for unwinding activity were not eliminated by these experiments.To further investigate this, an ATPase-deficient mutant enzyme was prepared that was unable to unwind DNA. If oligomerization were required for function, then the mutant enzyme would be expected to lower the activity of the wild type due to formation of hetero-oligomers. The effects of an ATPase-deficient mutant Dda protein (K38A Dda) on the activities of WT Dda were examined. The presence of K38A Dda protein, which is inactive as a helicase, but is still capable of binding DNA (Fig. 4), fails to decrease the ATPase activity of WT Dda (Fig. 5). The rate of unwinding under single cycle conditions was not reduced by the addition of the mutant enzyme, indicating that hetero-oligomers do not likely participate in the unwinding reaction. The amplitude for unwinding by WT Dda helicase was reduced upon addition of K38A Dda (Fig. 6). The data fall between that expected for a monomeric or dimeric helicase when analyzed as though a simple competition for substrate binding existed between wild type Dda and mutant or hetero-oligomeric Dda (Fig. 7 B). It is possible that more than one Dda monomer can bind to the substrate without invoking protein-protein interactions, which may lead to a greater than expected reduction in amplitude under conditions of excess enzyme and in the presence of the inactive K38A Dda.The effect of mixing K38A Dda and WT Dda was further investigated by conducting unwinding experiments in the presence of excess substrate. Under these conditions, the competition for DNA binding between the two proteins is effectively eliminated, because the protein will be distributed among the substrate molecules. If Dda is acting as a monomer, the addition of mutant protein should have no effect on WT Dda unwinding activity under these conditions because the mutant protein will bind to DNA, but will not prevent WT Dda binding and subsequent unwinding activity. However, if protein-protein interactions are required for unwinding, then such interactions should occur, despite the presence of excess DNA substrate. The results indicate that the presence of mutant Dda does not reduce the rate of unwinding (Fig.8 C), suggesting that Dda may not require oligomerization in order to function. Even when WT Dda alone or in the presence of K38A Dda is incubated with ATP prior to initiating the reaction with substrate, there is no reduction in the unwinding rate (Fig.8 C). Thus, Dda appears to be capable of functioning as a monomer. This may explain the fact that Dda is known to act in a distributive manner, cycling on and off of DNA during unwinding of long substrates (19Jongeneel C.V. Formosa T. Alberts B.M. J. Biol. Chem. 1984; 259: 12925-12932Abstract Full Text PDF PubMed Google Scholar). Perhaps this distributive mode of action is the result of a monomeric structure that does not allow Dda to encircle ssDNA in a manner similar to that of many hexameric helicases (4Egelman E. Curr. Biol. 1996; 4: 759-762Google Scholar, 18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar, 22Egelman E.H., Yu, X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (251) Google Scholar).In order to unwind a region of dsDNA, and translocate to a new site along the nucleic acid, helicases need two DNA binding sites so that the enzyme does not dissociate during unwinding. Therefore, Dda is expected to contain more than one site that is capable of binding to DNA. An inchworm mechanism has been suggested for unwinding and translocation by monomeric helicases. Evidence for multiple DNA binding sites on a monomeric helicase has been provided in the case of the PcrA helicase. The crystal structure of PcrA in the presence of a partial duplex DNA substrate indicates a binding site for ssDNA and an adjacent binding site for dsDNA (7Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). Dda is classified as a super family 1 helicase, like PcrA, based on sequence comparison (11Gorbalenya A. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1024) Google Scholar), although Dda is a 5′-to-3′ helicase (17Raney K.D. Benkovic S.J. J. Biol. Chem. 1995; 270: 22236-22242Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), whereas PcrA translocates 3′-to-5′ (30Dillingham M.S. Wigley D.B. Webb M.R. Biochemistry. 2000; 39: 205-212Crossref PubMed Scopus (197) Google Scholar).Another possible arrangement for "two" binding sites has been proposed for the NS3h helicase. The co-crystal structure of this enzyme in the presence of ssDNA indicates that 7 nucleotides are complexed within a groove between two adjacent domains and a third domain (31Kim J.L. Morgenstern J.P. Griffith J.P. Dwyer D.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure. 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). The two domains on one side of the groove were proposed to interact with the ssDNA such that one domain could move relative to the other as a function of ATP binding and hydrolysis. Hence, the ssDNA bound in one domain could be released during translocation while interactions in the second domain are maintained to hold onto the DNA. Whether one of the models suggested for PcrA or NS3h applies to Dda remains to be determined.Helicases in superfamily 1 and superfamily 2 have been difficult to characterize in terms of oligomeric structure, and much discussion has surrounded this issue. This is due to the fact that many biophysical approaches for studying oligomerization provide negative results when applied to these enzymes. Evidence has been presented that suggests that PcrA (7Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar), UvrD (8Mechanic L.E. Hall M.C. Matson S.W. J. Biol. Chem. 1999; 274: 12488-12498Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and NS3h (32Preugschat F. Danger D.P. Carter III, L.H. Davis R.G. Porter D.J.T. Biochemistry. 2000; 39: 5174-5183Crossref PubMed Scopus (28) Google Scholar) can function as monomers. Another helicase, PriA, was shown to exist as a monomer in solution, even when bound to ssDNA, although the functional form of the enzyme has not been determined (33Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2000; 275: 27865-27873Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Others have provided evidence that suggests that oligomeric forms of UvrD (34Ali J.A. Maluf N.K. Lohman T.M. J. Mol. Biol. 1999; 293: 815-834Crossref PubMed Scopus (93) Google Scholar) and NS3h (27Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) are required for optimal unwinding activity.Previously, the ability of Dda to displace streptavidin from the 3′ end of biotin-labeled oligonucleotides was found to exhibit a dependence on the length of the oligonucleotide. The streptavidin displacement reaction was found to proceed faster from longer oligonucleotides than from shorter ones (18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar). The results described here suggest that a monomeric form of Dda can function to unwind DNA. However, individual Dda monomers may align along the nucleic acid lattice to enhance overall activity in the streptavidin displacement assays, despite the lack of strong protein-protein interactions. When dsDNA1 is replicated, repaired, or recombined, the necessary ssDNA intermediates are provided by the activity of helicases (1Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (459) Google Scholar, 2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 3West S.C. Cell. 1996; 86: 177-180Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 4Egelman E. Curr. Biol. 1996; 4: 759-762Google Scholar, 5Matson S.W. Bean D.W. George J.W. Bioessays. 1994; 16: 13-22Crossref PubMed Scopus (269) Google Scholar). These enzymes appear to be ubiquitous, having been identified in viral, bacterial, and eukaryotic systems. Helicases hydrolyze nucleotide triphosphates, usually ATP, to obtain the energy needed to unwind dsDNA. They translocate on DNA often in a very processive manner, unwinding thousands of base pairs in a single binding event. Their processive nature implies that helicases have multiple DNA binding sites; indeed, a characteristic feature of many helicases is their propensity to form oligomeric structures, often dimers or hexamers. Knowledge of the oligomeric form of a helicase is of fundamental concern in development of a mechanism for unwinding activity, and many approaches have been applied to determine oligomeric structure. The lack of evidence for oligomerization from biophysical experiments has led to the suggestion that some helicases function as monomers. PcrA helicase of Bacillus stearothermophilus is proposed to function as a monomer that contains two binding sites for DNA (6Bird L.E. Brannigan J.A. Subramanya H.S. Wigley D.B. Nucleic Acids Res. 1998; 26: 2686-2693Crossref PubMed Scopus (91) Google Scholar, 7Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). Evidence has been provided that suggests Escherichia coli UvrD helicase (helicase II) can be active as a monomerin vivo and in vitro (8Mechanic L.E. Hall M.C. Matson S.W. J. Biol. Chem. 1999; 274: 12488-12498Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The E. coliRep helicase has been proposed to function as a dimer (2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar), whereas other helicases such as T7 gene 4 helicase and T4 gp41 helicase appear to function as hexamers (9Patel S.S. Hingorani M.M. J. Biol. Chem. 1993; 268: 10668-10675Abstract Full Text PDF PubMed Google Scholar, 10Dong F. Gogol E.P. von Hippel P. J. Biol. Chem. 1995; 270: 7462-7473Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Helicases have been classified according to sequence homology (11Gorbalenya A. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1024) Google Scholar), and those enzymes in superfamily 1 and superfamily 2 have proven difficult to characterize in terms of oligomerization. The focus of this study is the bacteriophage T4 helicase, Dda, a 5′-to-3′ helicase classified in superfamily 1. Evidence suggests that Dda is involved in T4 replication initiation (12Gauss P. Park K. Spencer T.E. Hacker K.J. J. Bacteriol. 1994; 176: 1667-1672Crossref PubMed Google Scholar). T4 Dda− mutants show a delayed DNA synthesis phenotype, consistent with this hypothesis. Dda also enhances the rate of branch migration owing to a specific interaction with the T4 recombinase (UvsX) and is therefore likely to play a role in recombination (13Kodadek T. Alberts B.M. Nature. 1987; 326: 312-314Crossref PubMed Scopus (52) Google Scholar, 14Hacker K.J. Alberts B.M. J. Biol. Chem. 1992; 267: 20674-20681Abstract Full Text PDF PubMed Google Scholar, 15Formosa T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (90) Google Scholar). Dda binds tightly to the T4 single-stranded DNA-binding protein, gp32, although the significance of this interaction has not been determined (15Formosa T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (90) Google Scholar, 16Jongeneel C.V. Bedinger P. Alberts B.M. J. Biol. Chem. 1984; 259: 12933-12938Abstract Full Text PDF PubMed Google Scholar). Dda translocates on ssDNA with a strong directional bias in the 5′-to-3′ direction (17Raney K.D. Benkovic S.J. J. Biol. Chem. 1995; 270: 22236-22242Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It is capable of removing protein blocks placed in the path of the enzyme, including streptavidin bound to biotin-labeled oligonucleotides (18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar). Dda is not highly processive (19Jongeneel C.V. Formosa T. Alberts B.M. J. Biol. Chem. 1984; 259: 12925-12932Abstract Full Text PDF PubMed Google Scholar,20Raney K.D. Sowers L.D. Millar D.P. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6644-6648Crossref PubMed Scopus (127) Google Scholar). Unwinding of only a few hundred base pairs can be prevented by addition of ssDNA, even when Dda is incubated with the substrate prior to addition of the competitor DNA (19Jongeneel C.V. Formosa T. Alberts B.M. J. Biol. Chem. 1984; 259: 12925-12932Abstract Full Text PDF PubMed Google Scholar). In contrast, the replicative helicase from bacteriophage T4, gp41, can unwind thousands of base pairs in a single binding event (21Dong F. Weitzel S.E. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14456-14461Crossref PubMed Scopus (44) Google Scholar). Like T7 gene 4 helicase, gp41 is a hexameric helicase that encircles ssDNA, resulting in very high processivity (18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar, 22Egelman E.H., Yu, X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (251) Google Scholar). The oligomeric nature of Dda was investigated in order to determine whether its relatively low processivity might be related to the oligomeric structure, and to lay the foundation for future mechanistic studies. DISCUSSIONThe search for a functional oligomeric structure of Dda has proven elusive. Experimental approaches such as chemical cross-linking and gel filtration did not strongly support nor eliminate formation of oligomeric species (Figs. 1 and 2). Biochemical assays in which the ATPase activity of Dda was measured at varying enzyme concentration did not provide any evidence for oligomerization (Fig. 3). Hence, methods that have provided support for oligomerization of other helicases gave negative results with Dda. Transient, protein-protein interactions that might be required for unwinding activity were not eliminated by these experiments.To further investigate this, an ATPase-deficient mutant enzyme was prepared that was unable to unwind DNA. If oligomerization were required for function, then the mutant enzyme would be expected to lower the activity of the wild type due to formation of hetero-oligomers. The effects of an ATPase-deficient mutant Dda protein (K38A Dda) on the activities of WT Dda were examined. The presence of K38A Dda protein, which is inactive as a helicase, but is still capable of binding DNA (Fig. 4), fails to decrease the ATPase activity of WT Dda (Fig. 5). The rate of unwinding under single cycle conditions was not reduced by the addition of the mutant enzyme, indicating that hetero-oligomers do not likely participate in the unwinding reaction. The amplitude for unwinding by WT Dda helicase was reduced upon addition of K38A Dda (Fig. 6). The data fall between that expected for a monomeric or dimeric helicase when analyzed as though a simple competition for substrate binding existed between wild type Dda and mutant or hetero-oligomeric Dda (Fig. 7 B). It is possible that more than one Dda monomer can bind to the substrate without invoking protein-protein interactions, which may lead to a greater than expected reduction in amplitude under conditions of excess enzyme and in the presence of the inactive K38A Dda.The effect of mixing K38A Dda and WT Dda was further investigated by conducting unwinding experiments in the presence of excess substrate. Under these conditions, the competition for DNA binding between the two proteins is effectively eliminated, because the protein will be distributed among the substrate molecules. If Dda is acting as a monomer, the addition of mutant protein should have no effect on WT Dda unwinding activity under these conditions because the mutant protein will bind to DNA, but will not prevent WT Dda binding and subsequent unwinding activity. However, if protein-protein interactions are required for unwinding, then such interactions should occur, despite the presence of excess DNA substrate. The results indicate that the presence of mutant Dda does not reduce the rate of unwinding (Fig.8 C), suggesting that Dda may not require oligomerization in order to function. Even when WT Dda alone or in the presence of K38A Dda is incubated with ATP prior to initiating the reaction with substrate, there is no reduction in the unwinding rate (Fig.8 C). Thus, Dda appears to be capable of functioning as a monomer. This may explain the fact that Dda is known to act in a distributive manner, cycling on and off of DNA during unwinding of long substrates (19Jongeneel C.V. Formosa T. Alberts B.M. J. Biol. Chem. 1984; 259: 12925-12932Abstract Full Text PDF PubMed Google Scholar). Perhaps this distributive mode of action is the result of a monomeric structure that does not allow Dda to encircle ssDNA in a manner similar to that of many hexameric helicases (4Egelman E. Curr. Biol. 1996; 4: 759-762Google Scholar, 18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar, 22Egelman E.H., Yu, X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (251) Google Scholar).In order to unwind a region of dsDNA, and translocate to a new site along the nucleic acid, helicases need two DNA binding sites so that the enzyme does not dissociate during unwinding. Therefore, Dda is expected to contain more than one site that is capable of binding to DNA. An inchworm mechanism has been suggested for unwinding and translocation by monomeric helicases. Evidence for multiple DNA binding sites on a monomeric helicase has been provided in the case of the PcrA helicase. The crystal structure of PcrA in the presence of a partial duplex DNA substrate indicates a binding site for ssDNA and an adjacent binding site for dsDNA (7Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). Dda is classified as a super family 1 helicase, like PcrA, based on sequence comparison (11Gorbalenya A. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1024) Google Scholar), although Dda is a 5′-to-3′ helicase (17Raney K.D. Benkovic S.J. J. Biol. Chem. 1995; 270: 22236-22242Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), whereas PcrA translocates 3′-to-5′ (30Dillingham M.S. Wigley D.B. Webb M.R. Biochemistry. 2000; 39: 205-212Crossref PubMed Scopus (197) Google Scholar).Another possible arrangement for "two" binding sites has been proposed for the NS3h helicase. The co-crystal structure of this enzyme in the presence of ssDNA indicates that 7 nucleotides are complexed within a groove between two adjacent domains and a third domain (31Kim J.L. Morgenstern J.P. Griffith J.P. Dwyer D.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure. 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). The two domains on one side of the groove were proposed to interact with the ssDNA such that one domain could move relative to the other as a function of ATP binding and hydrolysis. Hence, the ssDNA bound in one domain could be released during translocation while interactions in the second domain are maintained to hold onto the DNA. Whether one of the models suggested for PcrA or NS3h applies to Dda remains to be determined.Helicases in superfamily 1 and superfamily 2 have been difficult to characterize in terms of oligomeric structure, and much discussion has surrounded this issue. This is due to the fact that many biophysical approaches for studying oligomerization provide negative results when applied to these enzymes. Evidence has been presented that suggests that PcrA (7Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar), UvrD (8Mechanic L.E. Hall M.C. Matson S.W. J. Biol. Chem. 1999; 274: 12488-12498Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and NS3h (32Preugschat F. Danger D.P. Carter III, L.H. Davis R.G. Porter D.J.T. Biochemistry. 2000; 39: 5174-5183Crossref PubMed Scopus (28) Google Scholar) can function as monomers. Another helicase, PriA, was shown to exist as a monomer in solution, even when bound to ssDNA, although the functional form of the enzyme has not been determined (33Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2000; 275: 27865-27873Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Others have provided evidence that suggests that oligomeric forms of UvrD (34Ali J.A. Maluf N.K. Lohman T.M. J. Mol. Biol. 1999; 293: 815-834Crossref PubMed Scopus (93) Google Scholar) and NS3h (27Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) are required for optimal unwinding activity.Previously, the ability of Dda to displace streptavidin from the 3′ end of biotin-labeled oligonucleotides was found to exhibit a dependence on the length of the oligonucleotide. The streptavidin displacement reaction was found to proceed faster from longer oligonucleotides than from shorter ones (18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar). The results described here suggest that a monomeric form of Dda can function to unwind DNA. However, individual Dda monomers may align along the nucleic acid lattice to enhance overall activity in the streptavidin displacement assays, despite the lack of strong protein-protein interactions. The search for a functional oligomeric structure of Dda has proven elusive. Experimental approaches such as chemical cross-linking and gel filtration did not strongly support nor eliminate formation of oligomeric species (Figs. 1 and 2). Biochemical assays in which the ATPase activity of Dda was measured at varying enzyme concentration did not provide any evidence for oligomerization (Fig. 3). Hence, methods that have provided support for oligomerization of other helicases gave negative results with Dda. Transient, protein-protein interactions that might be required for unwinding activity were not eliminated by these experiments. To further investigate this, an ATPase-deficient mutant enzyme was prepared that was unable to unwind DNA. If oligomerization were required for function, then the mutant enzyme would be expected to lower the activity of the wild type due to formation of hetero-oligomers. The effects of an ATPase-deficient mutant Dda protein (K38A Dda) on the activities of WT Dda were examined. The presence of K38A Dda protein, which is inactive as a helicase, but is still capable of binding DNA (Fig. 4), fails to decrease the ATPase activity of WT Dda (Fig. 5). The rate of unwinding under single cycle conditions was not reduced by the addition of the mutant enzyme, indicating that hetero-oligomers do not likely participate in the unwinding reaction. The amplitude for unwinding by WT Dda helicase was reduced upon addition of K38A Dda (Fig. 6). The data fall between that expected for a monomeric or dimeric helicase when analyzed as though a simple competition for substrate binding existed between wild type Dda and mutant or hetero-oligomeric Dda (Fig. 7 B). It is possible that more than one Dda monomer can bind to the substrate without invoking protein-protein interactions, which may lead to a greater than expected reduction in amplitude under conditions of excess enzyme and in the presence of the inactive K38A Dda. The effect of mixing K38A Dda and WT Dda was further investigated by conducting unwinding experiments in the presence of excess substrate. Under these conditions, the competition for DNA binding between the two proteins is effectively eliminated, because the protein will be distributed among the substrate molecules. If Dda is acting as a monomer, the addition of mutant protein should have no effect on WT Dda unwinding activity under these conditions because the mutant protein will bind to DNA, but will not prevent WT Dda binding and subsequent unwinding activity. However, if protein-protein interactions are required for unwinding, then such interactions should occur, despite the presence of excess DNA substrate. The results indicate that the presence of mutant Dda does not reduce the rate of unwinding (Fig.8 C), suggesting that Dda may not require oligomerization in order to function. Even when WT Dda alone or in the presence of K38A Dda is incubated with ATP prior to initiating the reaction with substrate, there is no reduction in the unwinding rate (Fig.8 C). Thus, Dda appears to be capable of functioning as a monomer. This may explain the fact that Dda is known to act in a distributive manner, cycling on and off of DNA during unwinding of long substrates (19Jongeneel C.V. Formosa T. Alberts B.M. J. Biol. Chem. 1984; 259: 12925-12932Abstract Full Text PDF PubMed Google Scholar). Perhaps this distributive mode of action is the result of a monomeric structure that does not allow Dda to encircle ssDNA in a manner similar to that of many hexameric helicases (4Egelman E. Curr. Biol. 1996; 4: 759-762Google Scholar, 18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar, 22Egelman E.H., Yu, X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (251) Google Scholar). In order to unwind a region of dsDNA, and translocate to a new site along the nucleic acid, helicases need two DNA binding sites so that the enzyme does not dissociate during unwinding. Therefore, Dda is expected to contain more than one site that is capable of binding to DNA. An inchworm mechanism has been suggested for unwinding and translocation by monomeric helicases. Evidence for multiple DNA binding sites on a monomeric helicase has been provided in the case of the PcrA helicase. The crystal structure of PcrA in the presence of a partial duplex DNA substrate indicates a binding site for ssDNA and an adjacent binding site for dsDNA (7Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). Dda is classified as a super family 1 helicase, like PcrA, based on sequence comparison (11Gorbalenya A. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1024) Google Scholar), although Dda is a 5′-to-3′ helicase (17Raney K.D. Benkovic S.J. J. Biol. Chem. 1995; 270: 22236-22242Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), whereas PcrA translocates 3′-to-5′ (30Dillingham M.S. Wigley D.B. Webb M.R. Biochemistry. 2000; 39: 205-212Crossref PubMed Scopus (197) Google Scholar). Another possible arrangement for "two" binding sites has been proposed for the NS3h helicase. The co-crystal structure of this enzyme in the presence of ssDNA indicates that 7 nucleotides are complexed within a groove between two adjacent domains and a third domain (31Kim J.L. Morgenstern J.P. Griffith J.P. Dwyer D.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure. 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). The two domains on one side of the groove were proposed to interact with the ssDNA such that one domain could move relative to the other as a function of ATP binding and hydrolysis. Hence, the ssDNA bound in one domain could be released during translocation while interactions in the second domain are maintained to hold onto the DNA. Whether one of the models suggested for PcrA or NS3h applies to Dda remains to be determined. Helicases in superfamily 1 and superfamily 2 have been difficult to characterize in terms of oligomeric structure, and much discussion has surrounded this issue. This is due to the fact that many biophysical approaches for studying oligomerization provide negative results when applied to these enzymes. Evidence has been presented that suggests that PcrA (7Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar), UvrD (8Mechanic L.E. Hall M.C. Matson S.W. J. Biol. Chem. 1999; 274: 12488-12498Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and NS3h (32Preugschat F. Danger D.P. Carter III, L.H. Davis R.G. Porter D.J.T. Biochemistry. 2000; 39: 5174-5183Crossref PubMed Scopus (28) Google Scholar) can function as monomers. Another helicase, PriA, was shown to exist as a monomer in solution, even when bound to ssDNA, although the functional form of the enzyme has not been determined (33Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2000; 275: 27865-27873Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Others have provided evidence that suggests that oligomeric forms of UvrD (34Ali J.A. Maluf N.K. Lohman T.M. J. Mol. Biol. 1999; 293: 815-834Crossref PubMed Scopus (93) Google Scholar) and NS3h (27Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) are required for optimal unwinding activity. Previously, the ability of Dda to displace streptavidin from the 3′ end of biotin-labeled oligonucleotides was found to exhibit a dependence on the length of the oligonucleotide. The streptavidin displacement reaction was found to proceed faster from longer oligonucleotides than from shorter ones (18Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (112) Google Scholar). The results described here suggest that a monomeric form of Dda can function to unwind DNA. However, individual Dda monomers may align along the nucleic acid lattice to enhance overall activity in the streptavidin displacement assays, despite the lack of strong protein-protein interactions. We thank Dr. Barry Hurlburt for critical review of this manuscript.
The neat reaction of triethyl aluminum with triethyl phosphate yields a low molecular weight oil. The oligomer in an organic solvent was applied by spin coating onto porous alumina disks (Anodisc{reg_sign}, 0.02 micron filter) and calcined at 500{degrees}C to form amorphous AlPO{sub 4} membrane films. Alternatively, the films may be converted to crystalline molecular sieve films by hydrothermal treatment under pressure. The thickness and quality of the supported membranes were measured by scanning electron microscopy. The utility of the membranes for gas separations was determined by gas permeability measurements. The chemistry of the film precursor and properties of the supported membranes will be presented.
