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• W2063347630 abstract "During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the αH and αI helices in the thumb subdomain play in specific RNase H cleavage at the 3′-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3′-end. Further analysis of αH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage. During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the αH and αI helices in the thumb subdomain play in specific RNase H cleavage at the 3′-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3′-end. Further analysis of αH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage. reverse transcriptase human immunodeficiency virus type 1 polypurine tract murine leukemia virus primer-template nucleotide(s) minor groove binding track wild type The virus-encoded enzyme reverse transcriptase (RT)1 of human immunodeficiency virus type 1 (HIV-1) and other retroviruses catalyzes the conversion of genomic RNA to a double-stranded DNA replicative intermediate. As minus-strand DNA is synthesized, the RNA template is degraded by the RNase H activity of RT, which cleaves the RNA strand in an RNA-DNA hybrid (Refs. 1Collett M.S. Dierks P. Parsons J.T. Faras A.J. Nature. 1978; 272: 181-184Crossref PubMed Scopus (22) Google Scholar and 2Gilboa E. Mitra S.W. Goff S. Baltimore D. Cell. 1979; 18: 93-100Abstract Full Text PDF PubMed Scopus (420) Google Scholar; for reviews, see Refs. 3Katz R.A. Skalka A.M. Annu. Rev. Biochem. 1994; 63: 133-173Crossref PubMed Scopus (533) Google Scholar, 4Arts E.J. Wainberg M.A. Adv. Virus Res. 1996; 46: 97-163Crossref PubMed Google Scholar, 5Telesnitsky A. Goff S.P. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 121-160Google Scholar). This results in the production of many small RNA fragments, any one of which could potentially serve as a primer to initiate synthesis of plus-strand DNA (6Champoux J.J. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 103-117Google Scholar). However, a short, purine-rich sequence known as the polypurine tract (PPT) is almost exclusively used as the primer for plus-strand initiation (Refs. 7Huber H.E. Richardson C.C. J. Biol Chem. 1990; 265: 10565-10573Abstract Full Text PDF PubMed Google Scholar, 8Fuentes G.M. Rodrı́guez-Rodrı́guez L. Fay P.J. Bambara R.A. J. Biol. Chem. 1995; 270: 28169-28176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Randolph C.A. Champoux J.J. J. Biol. Chem. 1994; 269: 19207-19215Abstract Full Text PDF PubMed Google Scholar, 10Miller M.D. Wang B. Bushman F.D. J. Virol. 1995; 69: 3938-3944Crossref PubMed Google Scholar, 11Bowman E.H. Pathak V.K. Hu W.-S. J. Virol. 1996; 70: 1687-1694Crossref PubMed Google Scholar, 12Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar, 61Klarmann G.J. Yu H. Chen X. Dougherty J.P. Preston B.D. J. Virol. 1997; 71: 9259-9269Crossref PubMed Google Scholar; for a review, see Ref. 6Champoux J.J. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 103-117Google Scholar). Why the PPT sequence is selected from all of the other available primers has been the subject of much speculation.One possibility is that the PPT sequence is intrinsically resistant to RNase H degradation and therefore survives as the sole RNA primer available for plus-strand initiation. However, experimental evidence from HIV-1 (13Wöhrl B.M. Moelling K. Biochemistry. 1990; 29: 10141-10147Crossref PubMed Scopus (112) Google Scholar, 14Wöhrl B.M. Volkmann S. Moelling K. J. Mol. Biol. 1991; 220: 801-818Crossref PubMed Scopus (35) Google Scholar, 15Gao H.-Q. Boyer P.L. Arnold E. Hughes S.H. J. Mol. Biol. 1998; 277: 559-572Crossref PubMed Scopus (67) Google Scholar) 2K. Post and J. G. Levin, unpublished observations. 2K. Post and J. G. Levin, unpublished observations. and murine leukemia virus (MuLV) (9Randolph C.A. Champoux J.J. J. Biol. Chem. 1994; 269: 19207-19215Abstract Full Text PDF PubMed Google Scholar, 16Luo G. Sharmeen L. Taylor J. J. Virol. 1990; 64: 592-597Crossref PubMed Google Scholar, 17Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar) model systems demonstrates that cleavages within the PPT can occur. The MuLV PPT can also be internally cleaved by the isolated RNase H domain from MuLV RT; furthermore, the specificity of cleavage at the 3′-end of the PPT is lost when the polymerase domain is removed (18Schultz S.J. Champoux J.J. J. Virol. 1996; 70: 8630-8638Crossref PubMed Google Scholar, 19Zhan X. Crouch R.J. J. Biol. Chem. 1997; 272: 22023-22029Crossref PubMed Scopus (33) Google Scholar). Finally,Escherichia coli RNase H catalyzes cleavages within the MuLV PPT (9Randolph C.A. Champoux J.J. J. Biol. Chem. 1994; 269: 19207-19215Abstract Full Text PDF PubMed Google Scholar, 16Luo G. Sharmeen L. Taylor J. J. Virol. 1990; 64: 592-597Crossref PubMed Google Scholar, 17Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar, 19Zhan X. Crouch R.J. J. Biol. Chem. 1997; 272: 22023-22029Crossref PubMed Scopus (33) Google Scholar) as well as within the HIV-1 PPT. 3K. Post, M. D. Powell, and J. G. Levin, unpublished observations. 3K. Post, M. D. Powell, and J. G. Levin, unpublished observations.A second possibility to explain selection of the PPT primer is related to its unique helical structure (12Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar, 20Fedoroff O.Y. Ge Y. Reid B.R. J. Mol. Biol. 1997; 269: 225-239Crossref PubMed Scopus (62) Google Scholar) and the shape and width of the major groove, which is wider than that of other RNA-DNA hybrids (20Fedoroff O.Y. Ge Y. Reid B.R. J. Mol. Biol. 1997; 269: 225-239Crossref PubMed Scopus (62) Google Scholar,21Fedoroff O.Y. Salazar M. Reid B.R. J. Mol. Biol. 1993; 233: 509-523Crossref PubMed Scopus (234) Google Scholar). These structural factors could cause binding of RT to the PPT sequence to differ from the way RT binds to other primer-templates (P/T), thereby precluding cleavage within the PPT. Binding of RT to short RNA primers annealed to longer DNA templates has been suggested to occur through interaction of the polymerase active site with the 5′-end of the RNA (Fig. 1, IA; Ref. 22DeStefano J.J. Nucleic Acids Res. 1995; 23: 3901-3908Crossref PubMed Scopus (39) Google Scholar; for a review, see Ref. 23Hughes S.H. Hostomsky Z. Le Grice S.F.J. Lentz K. Arnold E. J. Virol. 1996; 70: 2679-2683Crossref PubMed Google Scholar). In this binding configuration, RNase H cleavage can occur; however, polymerization cannot. Interaction with the polymerase domain will direct RNase H cleavage to a site 14–18 nucleotides (nt) from the bound 5′-end, (14–18 nt being the distance between the polymerase and RNase H active sites (17Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar, 24Furfine E.S. Reardon J.E. J. Biol. Chem. 1991; 266: 406-412Abstract Full Text PDF PubMed Google Scholar, 25Fu T.-B. Taylor J. J. Virol. 1992; 66: 4271-4278Crossref PubMed Google Scholar, 26Gopalakrishnan V. Peliska J.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10763-10767Crossref PubMed Scopus (173) Google Scholar, 27Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar, 28Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1117) Google Scholar, 29Post K. Guo J. Kalman E. Uchida T. Crouch R.J. Levin J.G. Biochemistry. 1993; 32: 5508-5517Crossref PubMed Scopus (34) Google Scholar, 30Ben-Artzi H. Zeelon E. Amit B. Wortzel A. Gorecki M. Panet A. J. Biol. Chem. 1993; 268: 16465-16471Abstract Full Text PDF PubMed Google Scholar, 31Götte M. Fackler S. Hermann T. Perola E. Cellai L. Gross H.J. Le Grice S.F.J. Heumann H. EMBO J. 1995; 14: 833-841Crossref PubMed Scopus (80) Google Scholar).In the case of PPT-containing sequences, it appears that the 3′-end of the RNA primer is bound to the polymerase active site (Fig. 1,IB) and that extension is favored over RNase H cleavage (32Palaniappan C. Fuentes G.M. Rodrı́guez-Rodrı́guez L. Fay P.J. Bambara R.A. J. Biol. Chem. 1996; 271: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,33Palaniappan C. Kim J.K. Wisniewski M. Fay P.J. Bambara R.A. J. Biol. Chem. 1998; 273: 3808-3816Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This suggests a model in which non-PPT-containing fragments are preferentially degraded, while PPT-containing fragments are preferentially extended (Fig. 1, IB; Ref. 33Palaniappan C. Kim J.K. Wisniewski M. Fay P.J. Bambara R.A. J. Biol. Chem. 1998; 273: 3808-3816Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These conclusions also imply that RT interacts with the PPT in a highly specific manner and that the polymerase domain is mainly responsible for this specificity.In HIV-1 RT, two regions within the p66 subunit contribute substantially to the binding and positioning of the P/T (28Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1117) Google Scholar). The first region, known as the “primer grip,” comprises residues in the β12-β13 hairpin in the palm subdomain (Fig.2; Ref. 28Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1117) Google Scholar). As we demonstrated earlier, primer grip mutations have a profound effect on the ability of RT to extend the PPT and minus-strand RNA primers but little or no effect on the extension of DNA versions of the same primers (37Ghosh M. Williams J. Powell M.D. Levin J.G. Le Grice S.F.J. Biochemistry. 1997; 36: 5758-5768Crossref PubMed Scopus (52) Google Scholar, 38Powell M.D. Ghosh M. Jacques P.S. Howard K.J. Le Grice S.F.J. Levin J.G. J. Biol. Chem. 1997; 272: 13262-13269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Differences in RNA and DNA primer usage by RT are likely to result from differences in the helical structures of hybrids having RNA or DNA in the primer strand (20Fedoroff O.Y. Ge Y. Reid B.R. J. Mol. Biol. 1997; 269: 225-239Crossref PubMed Scopus (62) Google Scholar, 38Powell M.D. Ghosh M. Jacques P.S. Howard K.J. Le Grice S.F.J. Levin J.G. J. Biol. Chem. 1997; 272: 13262-13269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 39Yusupova G. Lanchy J.-M. Yusupov M. Keith G. Le Grice S.F.J. Ehresmann C. Ehresmann B. Marquet R. J. Mol. Biol. 1996; 261: 315-321Crossref PubMed Scopus (18) Google Scholar). Mutations in the primer grip region can also affect RNase H function (37Ghosh M. Williams J. Powell M.D. Levin J.G. Le Grice S.F.J. Biochemistry. 1997; 36: 5758-5768Crossref PubMed Scopus (52) Google Scholar, 38Powell M.D. Ghosh M. Jacques P.S. Howard K.J. Le Grice S.F.J. Levin J.G. J. Biol. Chem. 1997; 272: 13262-13269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 40Palaniappan C. Wisniewski M. Jacques P.S. Le Grice S.F.J. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 11157-11164Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar); for example, mutant Y232A is defective in utilization of an RNA primer and also exhibits altered cleavage specificity at the PPT (38Powell M.D. Ghosh M. Jacques P.S. Howard K.J. Le Grice S.F.J. Levin J.G. J. Biol. Chem. 1997; 272: 13262-13269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).Figure 2Interactions of the αH helix with the DNA minor groove. A view looking down the αH helix shows that this helix is embedded in the DNA minor groove and interacts primarily with the primer strand (white), while the αI helix sits over the template strand (blue). These interactions occur 3–9 nt upstream of the polymerase active site. The polymerase active site carboxylates (Asp110, Asp185, and Asp186) are illustrated in a ball-and-stick representation and denote the 3′ primer terminus. Primer grip residues are located in the loop between β-strands 12 and 13. The core residues of the αH helix (Gly262, Lys263, Asn265, and Trp266) are indicated. Gly262 is situated directly behind Trp266. Changes in these residues have a dramatic effect on RNase H cleavage specificity. This figure was made with MOLSCRIPT (34Kraulis P. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Raster3D (35Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3869) Google Scholar) with the coordinates from the HIV-1 RT-double-stranded DNA-Fab complex (36Ding J. Das K. Hsiou Y. Sarafianos S.G. Clark Jr., A.D. Jacobo-Molina A. Tantillo C. Hughes S.H. Arnold E. J. Mol. Biol. 1998; 284: 1095-1111Crossref PubMed Scopus (302) Google Scholar).View Large Image Figure ViewerDownload (PPT)A second region having a major role in binding of P/T is composed of portions of the antiparallel helices, αH and αI, in the p66 thumb subdomain (Fig. 2). It has been proposed that αH and αI residues form contacts that are important for holding the P/T in position during the translocation step in polymerization, and these residues have been collectively described as a “helix clamp” (41Hermann T. Meier T. Götte M. Heumann H. Nucleic Acids Res. 1994; 22: 4625-4633Crossref PubMed Scopus (45) Google Scholar, 42Hermann T. Heumann H. Eur. J. Biochem. 1996; 242: 98-103Crossref PubMed Scopus (11) Google Scholar). A refinement of the structure of the complex of HIV-1 RT with a double-stranded DNA P/T and the Fab fragment of a monoclonal antibody revealed the participation of αH and αI in a “translocation track” for bound P/T but showed no direct evidence of interactions in the minor groove (36Ding J. Das K. Hsiou Y. Sarafianos S.G. Clark Jr., A.D. Jacobo-Molina A. Tantillo C. Hughes S.H. Arnold E. J. Mol. Biol. 1998; 284: 1095-1111Crossref PubMed Scopus (302) Google Scholar).Functional analysis in combination with molecular dynamics modeling performed in an earlier study (43Bebenek K. Beard W.A. Darden T.A. Li L. Prasad R. Luxon B.A. Gorenstein D.G. Wilson S.H. Kunkel T.A. Nat. Struct. Biol. 1997; 3: 194-197Crossref Scopus (113) Google Scholar) led to the identification of a track-like element in p66 consisting of five amino acids: Gln258, Gly262, and Trp266 in the αH helix in the thumb subdomain; Gln269, just outside of αH; and Ile94, in the palm subdomain. This motif, termed the minor groove binding track (MGBT), interacts in the minor groove over a distance from the second to sixth base pair from the 3′ terminus of the primer (43Bebenek K. Beard W.A. Darden T.A. Li L. Prasad R. Luxon B.A. Gorenstein D.G. Wilson S.H. Kunkel T.A. Nat. Struct. Biol. 1997; 3: 194-197Crossref Scopus (113) Google Scholar) in the region where a bend of about 41° occurs in the bound P/T and the DNA undergoes a transition from A- to B-form helical structure (28Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1117) Google Scholar, 36Ding J. Das K. Hsiou Y. Sarafianos S.G. Clark Jr., A.D. Jacobo-Molina A. Tantillo C. Hughes S.H. Arnold E. J. Mol. Biol. 1998; 284: 1095-1111Crossref PubMed Scopus (302) Google Scholar). More recently, analysis of the x-ray crystal structure of a covalently trapped catalytic complex showed that site-specific cysteine mutations introduced into α-helix H residues Gln258, Gly262, and Trp266 could form specific cross-links with a single tethered thiol group placed in the minor groove of the bound double-stranded DNA substrate (44Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1354) Google Scholar); these findings are consistent with the MGBT proposal.In previous work, we conducted an extensive analysis of the effect of alanine-scanning mutations in α-helices H and I on polymerase activity and examined P/T binding, fidelity, and enzyme kinetics (43Bebenek K. Beard W.A. Darden T.A. Li L. Prasad R. Luxon B.A. Gorenstein D.G. Wilson S.H. Kunkel T.A. Nat. Struct. Biol. 1997; 3: 194-197Crossref Scopus (113) Google Scholar,45Beard W.A. Stahl S.J. Kim H.-R. Bebenek K. Kumar A. Strub M.-P. Becerra S.P. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1994; 269: 28091-28097Abstract Full Text PDF PubMed Google Scholar, 46Beard W.A. Minnick D.T. Wade C.L. Prasad R. Won R.L. Kumar A. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1996; 271: 12213-12220Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 47Bebenek K. Beard W.A. Casas-Finet J.R. Kim H.-R. Darden T.A. Wilson S.H. Kunkel T.A. J. Biol. Chem. 1995; 270: 19516-19523Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). This analysis showed that changes in individual residues of αI do not affect P/T binding or fidelity, although a reduced amount of active enzyme in the mutant preparations was noted (46Beard W.A. Minnick D.T. Wade C.L. Prasad R. Won R.L. Kumar A. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1996; 271: 12213-12220Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In contrast, a number of αH mutants, and especially G262A and W266A in the MGBT, exhibited lower binding affinity, processivity, and frameshift fidelity (43Bebenek K. Beard W.A. Darden T.A. Li L. Prasad R. Luxon B.A. Gorenstein D.G. Wilson S.H. Kunkel T.A. Nat. Struct. Biol. 1997; 3: 194-197Crossref Scopus (113) Google Scholar, 45Beard W.A. Stahl S.J. Kim H.-R. Bebenek K. Kumar A. Strub M.-P. Becerra S.P. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1994; 269: 28091-28097Abstract Full Text PDF PubMed Google Scholar, 47Bebenek K. Beard W.A. Casas-Finet J.R. Kim H.-R. Darden T.A. Wilson S.H. Kunkel T.A. J. Biol. Chem. 1995; 270: 19516-19523Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). A detailed investigation of mutants with substitutions other than alanine for Trp266 indicated that there is a strong correlation between buried apolar side-chain surface area and quantitative changes in P/T binding; it was suggested that both hydrophobic interactions and hydrogen bonding contribute to the stability of the RT-DNA complex (48Beard W.A. Bebenek K. Darden T.A. Li L. Prasad R. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1998; 273: 30435-30442Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Other studies have shown that mutations in the thumb subdomain can have a dramatic effect on the polymerase, RNase H, and minus-strand DNA transfer activities of HIV-1 RT (15Gao H.-Q. Boyer P.L. Arnold E. Hughes S.H. J. Mol. Biol. 1998; 277: 559-572Crossref PubMed Scopus (67) Google Scholar). RNase H assays with HIV-1 PPT-containing substrates consisting of a short DNA primer annealed to a longer RNA template revealed that the cleavage activities of wild-type (WT) RT and two αH mutants, W266T and G262A, differ, indicating that mutations in the thumb subdomain can affect the efficiency and specificity of RNase H cleavage (15Gao H.-Q. Boyer P.L. Arnold E. Hughes S.H. J. Mol. Biol. 1998; 277: 559-572Crossref PubMed Scopus (67) Google Scholar).In the present study, we have continued our analysis of alanine-scanning mutations in α-helices H and I but have changed the focus of our investigation from polymerase activity to RNase H cleavage. Our goal was to determine the effect that these mutations might have on positioning of the RNase H domain for correct removal of the PPT primer from nascent plus-strand DNA. We find that individual alanine substitutions at multiple positions in the αH and αI helices, as well as the alternate substitutions at position 266, result in a loss of cleavage specificity with PPT-containing substrates. Further, the effects appear to be specific for PPT cleavage rather than more general RNase H-catalyzed cleavage.DISCUSSIONIn the present study, we have investigated the role that residues in the αH and αI helices in the HIV-1 RT thumb subdomain play in positioning the bound P/T for correct removal of the PPT. Precise recognition and cleavage of the PPT is a crucial event in retrovirus replication and ultimately defines one end of proviral DNA (for reviews, see Refs. 6Champoux J.J. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 103-117Google Scholar, 59Varmus H. Brown P. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington, D. C.1989: 53-108Google Scholar, and 60Brown P.O. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 161-203Google Scholar). Our approach was to test RNase H-catalyzed primer removal activity of mutant enzymes bearing single alanine substitutions in residues 253–270 and 277–287 (45Beard W.A. Stahl S.J. Kim H.-R. Bebenek K. Kumar A. Strub M.-P. Becerra S.P. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1994; 269: 28091-28097Abstract Full Text PDF PubMed Google Scholar, 46Beard W.A. Minnick D.T. Wade C.L. Prasad R. Won R.L. Kumar A. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1996; 271: 12213-12220Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and single amino acid replacements of Trp266 (48Beard W.A. Bebenek K. Darden T.A. Li L. Prasad R. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1998; 273: 30435-30442Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). An in vitro assay was used that makes it possible to look specifically at PPT primer removal without regard to the polymerase activity of each enzyme (Fig. 3; Ref. 38Powell M.D. Ghosh M. Jacques P.S. Howard K.J. Le Grice S.F.J. Levin J.G. J. Biol. Chem. 1997; 272: 13262-13269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).Recently, a report by Gao et al. (15Gao H.-Q. Boyer P.L. Arnold E. Hughes S.H. J. Mol. Biol. 1998; 277: 559-572Crossref PubMed Scopus (67) Google Scholar) appeared that determined the effect of two point mutations in αH, i.e.G262A and W266T, on RNase H cleavage of PPT-containing substrates. The results of this study are not directly comparable with ours, since the substrates used were quite different; in their case, a 20-nt DNA primer, consisting of sequences including or near the PPT, was annealed to an 81-nt RNA template. In this configuration, RNase H cleavage is dictated by the 3′ terminus of the DNA primer (13Wöhrl B.M. Moelling K. Biochemistry. 1990; 29: 10141-10147Crossref PubMed Scopus (112) Google Scholar, 17Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar, 24Furfine E.S. Reardon J.E. J. Biol. Chem. 1991; 266: 406-412Abstract Full Text PDF PubMed Google Scholar, 25Fu T.-B. Taylor J. J. Virol. 1992; 66: 4271-4278Crossref PubMed Google Scholar, 26Gopalakrishnan V. Peliska J.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10763-10767Crossref PubMed Scopus (173) Google Scholar, 27Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar, 28Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1117) Google Scholar, 29Post K. Guo J. Kalman E. Uchida T. Crouch R.J. Levin J.G. Biochemistry. 1993; 32: 5508-5517Crossref PubMed Scopus (34) Google Scholar, 30Ben-Artzi H. Zeelon E. Amit B. Wortzel A. Gorecki M. Panet A. J. Biol. Chem. 1993; 268: 16465-16471Abstract Full Text PDF PubMed Google Scholar, 31Götte M. Fackler S. Hermann T. Perola E. Cellai L. Gross H.J. Le Grice S.F.J. Heumann H. EMBO J. 1995; 14: 833-841Crossref PubMed Scopus (80) Google Scholar). By contrast, the PPT-containing substrates employed here were all short RNA primers annealed to a longer DNA template and were similar to intermediates expected to form during the course of reverse transcription. Thus, the types of cleavage patterns generated in the two studies are qualitatively different. Nevertheless, despite these differences, the overall conclusion of both studies is the same, namely that mutations in αH affect the specificity of RNase H cleavage. Interestingly, based on assays with RTs having the W266T mutation in either the p66 or p51 subunit, Gao et al. (15Gao H.-Q. Boyer P.L. 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For this to occur, the 5′ terminus of the PPT must somehow “slip” from its normal binding position (Fig." @default.
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