Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5′-AC unpaired dinucleotide. These short-flap(pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5′-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5′-flaps from short- and long-flap oligonucleotide substrates that mimicpre- andpost-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5′-flaps by FEN-1 inpre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5′-flaps by FEN-1 frompost-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5′-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen withpre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall,post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase thanpre-repair structures. The site of cleavage was 1 or 2 nucleotides 3′ of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5′-flaps from eitherpre- orpost-repair substrates.
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Acel A, Udashkin BE, Wainberg M, Faust EA. Efficient gap repair catalyzed in vitro by an intrinsic DNA polymerase activity of human immunodeficiency virus type 1 integrase. J Virol 72:2062–2071;1998.
Alleva JL, Doetsch PW. Characterization ofSchizosaccharomyces pombe Rad2 protein, a FEN-1 homolog. Nucleic Acids Res 26:3645–3650;1998.
Andrake MD, Skalka AM. Retroviral integrase, putting the pieces together. J Biol Chem 271:19633–19636;1996.
Asante-Appiah E, Skalka AM. Molecular mechanisms in retrovirus DNA integration. Antiviral Res 36:139–156;1997.
Asante-Appiah E, Skalka AM. HIV-1 integrase: Structural organization, conformational changes, and catalysis. Adv Virus Res 52:351–369;1999.
Balakrishnan M, Jonsson CB. Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions. J Virol 71:1025–1035;1997.
Barnes CJ, Wahl AF, Shen B, Park MS, Bambara RA. Mechanism of tracking and cleavage of adduct-damaged DNA substrates by the mammalian 5′-to 3′-exonuclease/endonuclease RAD2 homologue 1 or flap endonuclease 1. J Biol Chem 271:29624–29631;1996.
Brinn E, Yi J, Skalka AM, Leis J. Modeling the late steps in HIV-1 retroviral integrase-catalyzed DNA integration. J Biol Chem 275:39287–39295;2000.
Carteau S, Gorelick RJ, Bushman FD. Coupled integration of human immunodeficiency virus type 1 cDNA ends by purified integrase in vitro: Stimulation by the viral nucleocapsid protein. J Virol 73:6670–6679;1999.
Cherepanov P, Surratt D, Toelen J, Pluymers W, Griffith J, De Clercq E, Debyser Z. Activity of recombinant HIV-1 integrase on mini-HIV DNA. Nucleic Acids Res 27:2202–2210;1999.
Donzella GA, Leon O, Roth MJ. Implication of a central cysteine residue and the HHCC domain of Moloney murine leukemia virus integrase protein in functional multimerization. J Virol 72:1691–1698;1998.
Drelich M, Wilhelm R, Mous J. Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro. Virology 188:459–468;1992.
Dyda F, Hickman AB, Jenkins TM, Engelman A, Craigie R, Davies DR. Crystal structure of the catalytic domain of HIV-1 integrase: Similarity to other polynucleotidyl transferases. Science 266:1981–1986;1994.
Ellison V, Gerton J, Vincent KA, Brown PO. An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer. J Biol Chem 270:3320–3326;1995.
Engelman A. In vivo analysis of retroviral integrase structure and function. Adv Virus Res 52:411–426;1999.
Engelman A, Craigie R. Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro. J Virol 66:6361–6369;1992.
Engelman A, Mizuuchi K, Craigie R. HIV-1 DNA integration: Mechanism of viral DNA cleavage and DNA strand transfer. Cell 61:1211–1221;1991.
Esposito D, Craigie R. HIV integrase structure and function. Adv Virus Res 52:319–333;1999.
Faust EA, Acel A, Udashkin B, Wainberg MA. Human immunodeficiency virus type 1 integrase stabilizes a linearized HIV-1 LTR plasmid in vivo. Biochem Mol Biol Int 36:745–758;1995.
Faust EA, Garg A, Small L, Acel A, Wald R, Udashkin B. Enzymatic capability of HIS-tagged HIV-1 integrase using oligonucleotide disintegration substrates. J Biomed Sci 3:254–265;1996.
Gary R, Kim K, Cornelius HL, Park MS, Matsumoto Y. Proliferating cell nuclear antigen facilitates excision in long-patch base excision repair. J Biol Chem 274:4354–4363;1999.
Goldgur Y, Dyda F, Hickman AB, Jenkins TM, Craigie R, Davies DR. Three new structures of the core domain of HIV-1 integrase: An active site that binds magnesium. Proc Natl Acad Sci USA 95:9150–9154;1998.
Gomes XV, Burgers PM. Two modes of FEN1 binding to PCNA regulated by DNA. EMBO J 19:3811–3821;2000.
Hansen MST, Bushman FD. Human immunodeficiency virus type 2 preintegration complexes: Activities in vitro and response to inhibitors. J Virol 71:3351–3356;1997.
Harrington JJ, Lieber MR. The characterization of a mammalian DNA structure-specific endonuclease. EMBO J 13:1235–1246;1994.
Hindmarsh P, Leis J. Retroviral DNA integration. Microbiol Mol Biol Rev 63:1092–2172;1999.
Hindmarsh P, Ridky T, Reeves R, Andrake M, Skalka AM, Leis J. HMG protein family members stimulate human immunodeficiency virus type 1 and avian sarcoma virus concerted DNA integration in vitro. J Virol 73:2994–3003;1999.
Hosfield DJ, Frank G, Weng Y, Tainer JA, Shen B. Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1. J Biol Chem 273:27154–27161;1998.
Hosfield DJ, Mol CD, Shen B, Tainer JA. Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: Coupling DNA and PCNA binding to FEN-1 activity. Cell 95:135–146;1998.
Hwang KY, Baek K, Kim HY, Cho Y. The crystal structure of flap endonuclease-1 fromMethanococcus jannaschii. Nat Struct Biol 5:707–713;1998.
Kalpana GV, Reicin A, Cheng GS, Sorin M, Paik S, Goff SP. Isolation and characterization of an oligomerization-negative mutant of HIV-1 integrase. Virology 259:274–285;1999.
Kim K, Biade S, Matsumoto Y. Involvement of flap endonuclease 1 in base excision DNA repair. J Biol Chem 273:8842–8848;1998.
Kulkosky J, Katz RA, Merkel G, Skalka AM. Activities and substrate specificity of the evolutionarily conserved central domain of retroviral integrase. Virology 206:448–456;1995.
Lee SP, Han MK. Zinc stimulates Mg2+-dependent 3′-processing activity of human immunodeficiency virus type 1 integrase in vitro. Biochemistry 35:3837–3844;1996.
Li L, Farnet CM, Anderson WF, Bushman FD. Modulation of activity of Moloney murine leukemia virus preintegration complexes by host factors in vitro. J Virol 72:2125–2131;1998.
Lieber MR. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. Bioessays 19:233–240;1997.
Lyamichev V, Brow MAD, Dahlberg JE. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260:778–783;1993.
Matsumoto Y, Kim K, Hurwitz J, Gary R, Levin DS, Tomkinson AE, Park MS. Reconstitution of proliferating cell nuclear antigen-dependent repair of apurinic/apyrimidinic sites with purified human proteins. J Biol Chem 274:33703–33708;1999.
McCord M, Chiu R, Vora AC, Grandgenett DP. Retrovirus DNA termini bound by integrase communicate in trans for full-site integration in vitro. Virology 259:392–401;1999.
Miller MD, Farnet CM, Bushman FD. Human immunodeficiency virus type 1 preintegration complexes: Studies of organization and composition. J Virol 71:5382–5390;1997.
Murante RS, Huang L, Turchi JJ, Bambara RA. The calf 5′- to 3′-exonuclease is also an endonuclease with both activities dependent on primers annealed upstream of the point of cleavage. J Biol Chem 269:1191–1196;1994.
Nolan JP, Shen B, Park MS, Sklar LA. Kinetic analysis of human flap endonuclease-1 by flow cytometry. Biochemistry 35:11668–11676;1996.
Robins P, Pappin DJC, Wood RD, Lindahl T. Structural and functional homology between mammalian DNase IV and the 5′-nuclease domain ofEsherichia coli DNA polymerase I. J Biol Chem 269:28535–28538;1994.
Roe T, Chow SA, Brown PO. 3′-End processing and kinetics of 5′-end joining during retroviral integration in vivo. J Virol 71:1334–1340;1997.
Shen B, Nolan JP, Sklar LA, Park MS. Essential amino acids for substrate binding and catalysis of human flap endonuclease 1. J Biol Chem 271:9173–9176;1996.
Shen B, Nolan JP, Sklar LA, Park MS. Functional analysis of point mutations in human flap endonuclease-1 active site. Nucleic Acids Res 25:3332–3338;1997.
Sommers CH, Miller EJ, Dujon B, Prakash L, Prakash S. Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5′- to 3′-exonuclease required for lagging strand DNA synthesis in reconstituted systems. J Biol Chem 270:4193–4196;1995.
Tom S, Henricksen LA, Bambara RA. Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1. J Biol Chem 275:10498–10505;2000.
van den Ent FMI, Vos A, Plasterk RHA. Mutational scan of the human immunodeficiency virus type 2 integrase protein. J Virol 72:3916–3924;1998.
van Gent DC, Groeneger AAMO, Plasterk RHA. Mutational analysis of the integrase protein of human immunodeficiency virus type 2. Proc Natl Acad Sci USA 89:9598–9602;1992.
van Gent DC, Vink C, Groeneger AMO, Plasterk RHA. Complementation between HIV integrase proteins mutated in different domains. EMBO J 12:3261–3267;1993.
Vincent KA, Ellison V, Chow SA, Brown PO. Characterization of human immunodeficiency virus type 1 integrase expressed inEscherichia coli and analysis of variants with amino terminal mutations. J Virol 67:425–437;1993.
Vink C, Groeneger AAMO, Plasterk RHA. Identification of the catalytic and DNA binding region of the human immunodeficiency virus type I integrase protein. Nucleic Acids Res 21:1419–1425;1993.
Vink C, Plasterk RHA. The human immunodeficiency virus integrase protein. Trends Genet 9:433–437;1993.
Vink C, Yeheskiely E, van der Marel GA, van Boom JH, Plasterk RHA. Site-specific hydrolysis and alcoholysis of human immunodeficiency virus DNA termini mediated by the viral integrase protein. Nucleic Acids Res 19:6691–6698;1992.
Warbrick E. PCNA binding through a conserved motif. Bioessays 20:195–199;1998.
Wolfe AL, Felock PJ, Hastings JC, Blau CU, Hazuda DJ. The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates. J Virol 70:1424–1432;1996.
Wu X, Li J, Li X, Hsieh CL, Burgers PM, Lieber MR. Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA. Nucleic Acids Res 24:2036–2043;1996.
Wu X, Wilson TE, Lieber MR. A role for FEN-1 in nonhomologous DNA end joining: The order of strand annealing and nucleolytic processing events. Proc Natl Acad Sci USA 96:1303–1308;1999.
Yoder KE, Bushman FD. Repair of gaps in retroviral DNA integration intermediates. J Virol 74:11191–11200;2000.
Yoon JH, Swiderski PM, Kaplan BE, Takao M, Yasui A, Shen B, Pfeifer GP. Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway. Biochemistry 38:4809–4817;1999.
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Faust, E.A., Triller, H. Stimulation of human flap endonuclease 1 by human immunodeficiency virus type 1 integrase: Possible role for flap endonuclease 1 in 5′-end processing of human immunodeficiency virus type 1 integration intermediates. J Biomed Sci 9, 273–287 (2002). https://doi.org/10.1007/BF02256074
- DNA repair