Key points are not available for this paper at this time.
We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A′) prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, A. H., Griffin, K., Washington, M. T., Patel, S. S., and Studier, F. W. (1996) J. Biol. Chem. 271, 26819-26824). Mutants were selected from each of the helicase-conserved motifs for detailed analysis to understand better their function. In agreement with the in vivo results, the mutants were defective in helicase activity but were active in primase function. dTTP hydrolysis, DNA binding, and hexamer formation were examined. Three classes of defective mutants were observed. Group A mutants (E348K, D424N, and S496F), defective in dTTP hydrolysis, lie in motifs 1a, 2, and 4 and are possibly involved in NTP binding/hydrolysis. Group B mutants (R487C and G488D), defective in DNA binding, lie in motif 4 and are responsible directly or indirectly for DNA binding. Group C mutants (G116D, A257T, S345F, and G451E) were not defective in any of the activities except the helicase function. These mutants, scattered throughout the protein, appear defective in coupling dTTPase activity to helicase function. Secondary structural predictions of 4A′ and DnaB helicases resemble the known structures of RecA and F1-ATPase enzymes. Alignment shows a striking correlation in the positions of the amino acids that interact with NTP and DNA. We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A′) prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, A. H., Griffin, K., Washington, M. T., Patel, S. S., and Studier, F. W. (1996) J. Biol. Chem. 271, 26819-26824). Mutants were selected from each of the helicase-conserved motifs for detailed analysis to understand better their function. In agreement with the in vivo results, the mutants were defective in helicase activity but were active in primase function. dTTP hydrolysis, DNA binding, and hexamer formation were examined. Three classes of defective mutants were observed. Group A mutants (E348K, D424N, and S496F), defective in dTTP hydrolysis, lie in motifs 1a, 2, and 4 and are possibly involved in NTP binding/hydrolysis. Group B mutants (R487C and G488D), defective in DNA binding, lie in motif 4 and are responsible directly or indirectly for DNA binding. Group C mutants (G116D, A257T, S345F, and G451E) were not defective in any of the activities except the helicase function. These mutants, scattered throughout the protein, appear defective in coupling dTTPase activity to helicase function. Secondary structural predictions of 4A′ and DnaB helicases resemble the known structures of RecA and F1-ATPase enzymes. Alignment shows a striking correlation in the positions of the amino acids that interact with NTP and DNA. INTRODUCTIONDNA helicases catalyze unwinding of duplex DNA to single-stranded DNA, a process energetically coupled to NTP hydrolysis. Helicases are an important class of proteins required in almost all the processes of DNA and RNA metabolism. Recently, a large number of putative helicases have been identified mainly from amino acid sequence homologies. Known helicases have homologous amino acid sequences, confined to small regions in the protein, that are used as signature motifs for identifying helicases (1Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1021) Google Scholar, 2Ilyina T.V. Gorbalenya A.E. Koonin E.V. J. Mol. Evol. 1992; 34: 351-357Crossref PubMed Scopus (170) Google Scholar). Since a high resolution structure of a helicase is not known at the present time, the roles of these conserved motifs remain largely unclear.We are studying the mechanism of bacteriophage T7 DNA helicase, which is involved in DNA replication. Bacteriophage T7 is a model system used to study the detailed mechanisms of DNA replication because of its simplicity. A minimum of two proteins, T7 DNA polymerase and T7 DNA primase/helicase, have been shown to reconstitute duplex DNA replication in vitro. T7 gene 4 encodes the two primase/helicase proteins, 4A and 4B (3Dunn J.J. Studier F.W. J. Mol. Biol. 1981; 148: 303-330Crossref PubMed Scopus (126) Google Scholar). The full-length 63-kDa 4A protein has both helicase and primase activities, whereas the shorter 56-kDa 4B protein that begins at a second initiation codon has only helicase activity (4Bernstein J.A. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 396-400Crossref PubMed Scopus (93) Google Scholar, 5Bernstein J.A. Richardson C.C. J. Biol. Chem. 1989; 264: 13066-13073Abstract Full Text PDF PubMed Google Scholar). The helicase activity unwinds double-stranded DNA during leading strand DNA replication, and the primase catalyzes synthesis of tetraribonucleotides that serve as primers for lagging strand DNA replication (4Bernstein J.A. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 396-400Crossref PubMed Scopus (93) Google Scholar).T7 DNA helicase belongs to the general class of hexameric helicases. Its low resolution structure, studied in detail using electron microscopy and image averaging, shows that both 4A′ and 4B proteins form ring-shaped hexamers, and the ssDNA 1The abbreviations used are: ssDNAsingle-stranded DNAbpbase pair(s)bovine serum albumin DEAEdiethylaminoethyldTMP-PCPβ,γ-methylene deoxythymidine triphosphateDTTdithiothreitolPAGEpolyacrylamide gel electrophoresiskbkilobase pair(s). binds through the central hole of the ring (6Egelman 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). This mode of DNA binding results in protection of about 25 bases of ssDNA from nuclease digestion (7Hingorani M.M. Patel S.S. Biochemistry. 1993; 32: 12478-12487Crossref PubMed Scopus (102) Google Scholar) and likely confers high processivity to DNA unwinding. The helicase forms hexamers only in the presence of nucleotide ligands such as dTDP, dTTP, ATP, and dTMP-PCP (8Patel S.S. Hingorani M.M. J. Biol. Chem. 1993; 268: 10668-10675Abstract Full Text PDF PubMed Google Scholar, 9Hingorani M.M. Patel S.S. Biochemistry. 1996; 35: 2218-2228Crossref PubMed Scopus (68) Google Scholar). DNA binds tightly only to the hexameric species and requires the presence of dTTP or dTMP-PCP (7Hingorani M.M. Patel S.S. Biochemistry. 1993; 32: 12478-12487Crossref PubMed Scopus (102) Google Scholar). The various activities of the helicase protein such as NTP binding/hydrolysis, protein oligomerization, and DNA binding are linked (9Hingorani M.M. Patel S.S. Biochemistry. 1996; 35: 2218-2228Crossref PubMed Scopus (68) Google Scholar). Therefore, it is likely that amino acids responsible for these activities also may be close in space or perhaps lie in the same motif.Regions of T7 gene 4 protein show sequence homology to several bacterial and bacteriophage primase/helicase and primase-related helicases that belong to the DnaB family of helicases. Comparison of amino acid sequences in this family of helicases has led to the identification of five conserved motifs denoted 1A, 1a, 2B, 3, and 4 (2Ilyina T.V. Gorbalenya A.E. Koonin E.V. J. Mol. Evol. 1992; 34: 351-357Crossref PubMed Scopus (170) Google Scholar). Conserved motif 1A is the well known GXXGXGKT/S sequence found in numerous nucleotide-binding proteins and shown in many ATPases to be involved in binding the diphosphate or the triphosphate moiety of nucleotides (10Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4220) Google Scholar). Motif 2B is most likely the conserved motif B sequence involved in binding nucleotide via Mg2+ confirmed from this study. The remaining three motifs, 1a, 3, 4 have unknown functions.Site-directed mutagenesis has been used in the past to probe the function of several of the conserved motifs. Mutations have been made in the 1A motif, GXGKS sequence. Replacement of lysine 318 in this motif with an alanine (11Patel S.S. Hingorani M.M. Biochemistry. PubMed Scopus Google Scholar) and of with and lysine 318 with Richardson C.C. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) in of the dTTP activity and of the helicase mutagenesis motif and proteins in the of nucleotide Richardson C.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). These mutants also have dTTPase ssDNA binding, and duplex DNA unwinding the of this motif not the accompanying paper Patel S.S. Studier F.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google random mutagenesis and were used to mutants in the T7 primase/helicase gene The mutants selected were throughout of the protein, and most of primase function for T7 but were defective in helicase function. of the mutants in the of the protein lie in or close to conserved helicase motifs, and the mutants be for the function of these motifs. In this present the of nine of the duplex DNA, a helicase requires a number of NTP binding, NTP hydrolysis, oligomerization, DNA binding, and these various A in any of these to a in the unwinding We have used random mutagenesis and to mutants of T7 primase/helicase with the of identifying amino acids or regions of the protein in these various A detailed analysis of these mutants has to understand the for the helicase at the of these various mutagenesis a number of amino acid throughout the protein that in the helicase function required in vivo Patel S.S. Studier F.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of the mutants primase except that The of the nine proteins in this paper are with their in vivo all were to RNA primers but in helicase and bacteriophage both the helicase and primase activities be by the same protein and from amino acid sequence homology to and it that the of the protein all the primase motifs and the the helicase motifs. The two activities are coupled to a because amino acid in the the helicase activity and The mutants in the and that for ssDNA a in primase This that motif likely involved in DNA binding for the helicase may also be important for RNA The motif amino that the primase may not be the only DNA for primase (6Egelman 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) has shown that ssDNA binds through the central hole of the hexamer as shown in The of the DNA binds the of the hexamer the which have identified using as the or the helicase M. Washington, and S. S. Patel, in has been that a the of the primase is not for and a the of the primase is required Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We that the the is by the the hole of the hexamer the binds to the primase sequence This mode of DNA binding the primase activity of the mutants, and have DNA binding This may also the of primase by the DnaB helicase in and the of primase by the helicase in bacteriophage of the have nine mutants from both the and from the various conserved motifs of the C for their with to the various the nine 4A′ mutants have been three A characterized is also for Group A mutants are defective in dTTPase These mutants are confined to the and lie in the various conserved helicase motifs. Group B mutants are defective in DNA binding but in dTTPase These mutants are also confined to the Group C mutants are defective in coupling dTTPase and helicase These mutants have and the mutants are not confined to the but are found throughout the We have not identified any mutants defective in that the hexamer a large be required to to a of the of the 4A′ DNA mutants are of hexamer formation in the of ssDNA is their hexamer formation activity as by that the hexamer is present to the same as the the gel in the presence of that the hexamer is but it is not as as the in the presence of binding of ssDNA to protein in the binding binding binding. binding by the binding mutants are of hexamer formation in the of ssDNA is their hexamer formation activity as by that the hexamer is present to the same as the the gel in the presence of that the hexamer is but it is not as as the in the presence of binding of ssDNA to protein in the binding binding binding. binding by the binding in a A mutants defective in dTTP are found in helicase motifs. Mutants D424N, and lie motifs 1A, 1a, 2B, and 1A and 2B are the well known A and B sequences that have been shown in a number of proteins to be involved in NTP binding. acids in 1A form a that with the of NTP and Mg2+ in proteins such as and RecA M. R. 1993; PubMed Scopus Google Scholar, R. J.E. PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar). in motif 2B is found to interact with the NTP via the Mg2+ that binds to the of NTP R. J.E. PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar). In to these mutagenesis results show that of motif and motif 4 also in dTTP binding/hydrolysis, as mutants in motif and in motif 4 are defective in their dTTPase A mutants DNA, except which DNA binding in the presence of dTTP or The is from the mutants in that it shows a both in dTTPase and DNA binding. This is because mutants in motif 4 are also defective in DNA binding. the amino acids in motif 4 may a both in DNA binding and dTTP B mutants show a in DNA binding. have identified only two B mutants, and both lie in motif Mutants and were in binding and dTTP, hexamers by but these mutants DNA from three are with a DNA binding in these these mutants not DNA by gel DNA binding by a DNA binding these mutants have dTTPase not show dTTPase Since the DNA binding regions of helicases are not the that these amino acids in motif 4 may be indirectly involved in with the DNA. In these amino acids a in the for DNA binding that to and strand have shown that in T7 helicase, DNA binding is by dTTP binding and (7Hingorani M.M. Patel S.S. Biochemistry. 1993; 32: 12478-12487Crossref PubMed Scopus (102) Google Scholar). 4A′ protein binds DNA with a high in the presence of the of the of 4A′ for the DNA is in the presence of dTDP, the of dTTP hydrolysis. Since the of the nucleotide triphosphate or the for DNA, is a that the nucleotide-binding and the to the in such as M. R. 1993; PubMed Scopus Google Scholar). We that motif 4 involved in both dTTP and DNA binding may be of that that motif 4 is important for hexamer formation of three mutants, and Richardson C.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). is known that nucleotide binding, hexamer and DNA binding are all linked Therefore, the that this is involved in hexamer formation may be an of the function of the in this In the of this motif in hexamer formation is not with the of random mutants reported in this We have shown that the and mutants were not defective in In hexamers in the of DNA that are the that the reported also to have in but a in DNA binding. that these were to of the the hexamer Richardson C.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). this is the most of the three the that the and a in DNA binding. The of in this hexamer formation may be an because of the coupling DNA binding and hexamer C mutants not show a in any of the These mutants dTTP and DNA and the to the small DNA. We have these mutants as a in coupling the to helicase function. is not in helicases that in a in coupling nucleotide and DNA unwinding. Mutations coupling have been reported for S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and RNA helicase initiation A. EMBO J. 1992; PubMed Scopus Google Scholar). In initiation the mutants defective in coupling to helicase activity are found the A motif, B motif, and a conserved RNA C mutants are defective in DNA unwinding or have a in coupling the of nucleotide to DNA unwinding. of their large number and their throughout the protein in both it is likely that these mutants are defective in about by a in of the of activities through a of The dTTPase activity may be from activity for DNA unwinding. In such a the proteins dTTP but to the DNA, to the these mutants may be defective in This to of the protein which both and DNA unwinding. The two of may be by of dTTP binding DNA binding the same of the or the various all C mutants have a in the dTTPase activity to The dTTPase activity is the of or the for the of detailed be required to the for the and the dTTPase activity of this of of the C mutants is that helicase activity in the presence of the DNA with the This as these mutants were defective in the unwinding Since T7 DNA polymerase the helicase activity of these mutants that the polymerase the from the Since T7 primase/helicase protein forms a with T7 DNA polymerase Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. J.E. Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google it be the formation of this that of activity of the C The mechanism by which DNA polymerase the helicase activity of these mutants is of the helicase activity of C mutants by the polymerase is with that these mutants may be defective in because it is to the DNA polymerase the of the helicase that is defective in or processivity the activity of the defective in DNA unwinding strand of the C mutants in the presence of the polymerase is not with the in vivo results in the paper Patel S.S. Studier F.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The mutants are likely with the T7 polymerase in but to helicase activity to the T7 be a in the in and the in vivo in 4A′ which in the in is likely that in vivo T7 DNA polymerase is to the of these mutants to T7 or the mutants may be to function in processes during for initiation of DNA replication helicase activity has been shown to be important in Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google of the high resolution structure of a helicase is known at the present has been made to the of the mutants with to the by structure predictions of 4A′ and with the known structure of RecA 1992; PubMed Scopus Google Scholar) and F1-ATPase R. J.E. PubMed Scopus Google Scholar). F1-ATPase shows a of sequence homology to protein which is a hexameric protein that has unwinding activity J. Mol. Biol. 1995; Scopus Google Scholar). F1-ATPase and RecA proteins are to the 4A′ The F1-ATPase is a ring-shaped hexamer with nucleotide-binding and it with the of the the central of the in an to the mode of ssDNA binding to The RecA protein forms ssDNA which binds in the of the protein In the of RecA protein that interact with the DNA to the of the F1-ATPase that the J. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google structures of 4A′ and the DnaB protein were using from 1995; Google Scholar, PubMed Scopus Google Scholar). A structure from five PubMed Scopus Google Scholar, J. J. Mol. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, 1: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). sequence homology the F1-ATPase and the RecA protein, both structures are in their structures and R. J.E. PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar). The structural of 4A′ also well to that of F1-ATPase and the A and B nucleotide binding the of which the NTP and DNA binding motifs, has been with the known structure of the A motif and B motif have been used as for The is shown in The motif 1A begins with a and is by the the GXGKS sequence. Conserved motif as a and the The structural of 4A′ for these is a but for the DnaB protein, this is a that with the F1-ATPase The motif and motif 2B in F1-ATPase and 4A′ or DnaB shows the structural at conserved motif to that forms a a Conserved motif is a by a and a The F1-ATPase structure and the structure of 4A′ well also from to that motif The of to in the RecA structure and the in the F1-ATPase This structure is with the of the mutants in these conserved structure of 4A′ helicase The structure of 4A′ is with the known structure of the F1-ATPase as in the The the and of each structural and The helicase-conserved motifs are the 4A′ sequence. the 4A′ structure, the positions of A mutants D424N, and the of B mutants and the of the C mutants and the F1-ATPase structure, the of amino acid that interact with the nucleotide the the of amino acid that interact with the nucleotide the The of A and B nucleotide binding motifs and the are the A mutants D424N, and in nucleotide are in regions that to of the F1-ATPase protein that directly the are five regions in F1-ATPase that ATP, the A motif, a and a to conserved motif 1a, the B motif, the to motif 4 in helicase in F1-ATPase nucleotide to the in the hexameric and a at the C that the nucleotide is in A the is in the B is in motif and the is in the in motif to it is also that 4A′ may NTP at the This be with the that nucleotide binding is linked to hexamer such is the of the the in motif 4 in may also be involved in with the nucleotide to the of the structural of motif 4 as of the and the the nucleotide-binding and the The these two is the in the which the in the central of the ring-shaped and the in the RecA protein that with the DNA. INTRODUCTIONDNA helicases catalyze unwinding of duplex DNA to single-stranded DNA, a process energetically coupled to NTP hydrolysis. Helicases are an important class of proteins required in almost all the processes of DNA and RNA metabolism. Recently, a large number of putative helicases have been identified mainly from amino acid sequence homologies. Known helicases have homologous amino acid sequences, confined to small regions in the protein, that are used as signature motifs for identifying helicases (1Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1021) Google Scholar, 2Ilyina T.V. Gorbalenya A.E. Koonin E.V. J. Mol. Evol. 1992; 34: 351-357Crossref PubMed Scopus (170) Google Scholar). Since a high resolution structure of a helicase is not known at the present time, the roles of these conserved motifs remain largely unclear.We are studying the mechanism of bacteriophage T7 DNA helicase, which is involved in DNA replication. Bacteriophage T7 is a model system used to study the detailed mechanisms of DNA replication because of its simplicity. A minimum of two proteins, T7 DNA polymerase and T7 DNA primase/helicase, have been shown to reconstitute duplex DNA replication in vitro. T7 gene 4 encodes the two primase/helicase proteins, 4A and 4B (3Dunn J.J. Studier F.W. J. Mol. Biol. 1981; 148: 303-330Crossref PubMed Scopus (126) Google Scholar). The full-length 63-kDa 4A protein has both helicase and primase activities, whereas the shorter 56-kDa 4B protein that begins at a second initiation codon has only helicase activity (4Bernstein J.A. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 396-400Crossref PubMed Scopus (93) Google Scholar, 5Bernstein J.A. Richardson C.C. J. Biol. Chem. 1989; 264: 13066-13073Abstract Full Text PDF PubMed Google Scholar). The helicase activity unwinds double-stranded DNA during leading strand DNA replication, and the primase catalyzes synthesis of tetraribonucleotides that serve as primers for lagging strand DNA replication (4Bernstein J.A. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 396-400Crossref PubMed Scopus (93) Google Scholar).T7 DNA helicase belongs to the general class of hexameric helicases. Its low resolution structure, studied in detail using electron microscopy and image averaging, shows that both 4A′ and 4B proteins form ring-shaped hexamers, and the ssDNA 1The abbreviations used are: ssDNAsingle-stranded DNAbpbase pair(s)bovine serum albumin DEAEdiethylaminoethyldTMP-PCPβ,γ-methylene deoxythymidine triphosphateDTTdithiothreitolPAGEpolyacrylamide gel electrophoresiskbkilobase pair(s). binds through the central hole of the ring (6Egelman 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). This mode of DNA binding results in protection of about 25 bases of ssDNA from nuclease digestion (7Hingorani M.M. Patel S.S. Biochemistry. 1993; 32: 12478-12487Crossref PubMed Scopus (102) Google Scholar) and likely confers high processivity to DNA unwinding. The helicase forms hexamers only in the presence of nucleotide ligands such as dTDP, dTTP, ATP, and dTMP-PCP (8Patel S.S. Hingorani M.M. J. Biol. Chem. 1993; 268: 10668-10675Abstract Full Text PDF PubMed Google Scholar, 9Hingorani M.M. Patel S.S. Biochemistry. 1996; 35: 2218-2228Crossref PubMed Scopus (68) Google Scholar). DNA binds tightly only to the hexameric species and requires the presence of dTTP or dTMP-PCP (7Hingorani M.M. Patel S.S. Biochemistry. 1993; 32: 12478-12487Crossref PubMed Scopus (102) Google Scholar). The various activities of the helicase protein such as NTP binding/hydrolysis, protein oligomerization, and DNA binding are linked (9Hingorani M.M. Patel S.S. Biochemistry. 1996; 35: 2218-2228Crossref PubMed Scopus (68) Google Scholar). Therefore, it is likely that amino acids responsible for these activities also may be close in space or perhaps lie in the same motif.Regions of T7 gene 4 protein show sequence homology to several bacterial and bacteriophage primase/helicase and primase-related helicases that belong to the DnaB family of helicases. Comparison of amino acid sequences in this family of helicases has led to the identification of five conserved motifs denoted 1A, 1a, 2B, 3, and 4 (2Ilyina T.V. Gorbalenya A.E. Koonin E.V. J. Mol. Evol. 1992; 34: 351-357Crossref PubMed Scopus (170) Google Scholar). Conserved motif 1A is the well known GXXGXGKT/S sequence found in numerous nucleotide-binding proteins and shown in many ATPases to be involved in binding the diphosphate or the triphosphate moiety of nucleotides (10Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4220) Google Scholar). Motif 2B is most likely the conserved motif B sequence involved in binding nucleotide via Mg2+ confirmed from this study. The remaining three motifs, 1a, 3, 4 have unknown functions.Site-directed mutagenesis has been used in the past to probe the function of several of the conserved motifs. Mutations have been made in the 1A motif, GXGKS sequence. Replacement of lysine 318 in this motif with an alanine (11Patel S.S. Hingorani M.M. Biochemistry. PubMed Scopus Google Scholar) and of with and lysine 318 with Richardson C.C. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) in of the dTTP activity and of the helicase mutagenesis motif and proteins in the of nucleotide Richardson C.C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). These mutants also have dTTPase ssDNA binding, and duplex DNA unwinding the of this motif not the accompanying paper Patel S.S. Studier F.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google random mutagenesis and were used to mutants in the T7 primase/helicase gene The mutants selected were throughout of the protein, and most of primase function for T7 but were defective in helicase function. of the mutants in the of the protein lie in or close to conserved helicase motifs, and the mutants be for the function of these motifs. In this present the of nine of the
Washington et al. (Tue,) studied this question.