Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. and Harrison, S.C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These 'selectively deleted' heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Δ13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from pSI had little effect on synthesis-dependent and synthesis- independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Δ13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis

Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces

We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of ifiamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules

A clamp-like biohybrid catalyst for DNA oxidation,A bio-hybrid rotaxane catalyst

Contains fulltext : 117355.pdf (preprint version ) (Open Access) Contains fulltext : 117355_suppl.pdf (preprint version ) (Open Access

Truncating α-Helix E′ of p66 human immunodeficiency virus reverse transcriptase modulates RNase H function and impairs DNA strand transfer

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

Biohybrid DNA nuclease mimic

Item does not contain fulltex