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Truncating α-Helix E′ of p66 Human Immunodeficiency Virus Reverse Transcriptase Modulates RNase H Function and Impairs DNA Strand Transfer

1995; Elsevier BV; Volume: 270; Issue: 13 Linguagem: Inglês

10.1074/jbc.270.13.7068

1083-351X

Madhumita Ghosh, Kathryn J. Howard, Craig E. Cameron, Stephen J. Benkovic, Stephen H. Hughes, Stuart F.J. Le Grice,

DNA and Nucleic Acid Chemistry

The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed α-helix E′ of the RNase H domain (p66Δ8/p51 and p66Δ16/p51, respectively), while mutant p66Δ23/p51 lacked αE′ and the β5′-αE′ connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66Δ16/p51 and p66Δ23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66Δ8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66Δ8/p51 RT barely supported transfer of nascent(-)-strand DNA between RNA templates representing the 5′ and 3′ ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication. The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed α-helix E′ of the RNase H domain (p66Δ8/p51 and p66Δ16/p51, respectively), while mutant p66Δ23/p51 lacked αE′ and the β5′-αE′ connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66Δ16/p51 and p66Δ23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66Δ8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66Δ8/p51 RT barely supported transfer of nascent(-)-strand DNA between RNA templates representing the 5′ and 3′ ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication.