title: Substrate variations that affect the nucleic acid clamp activity of reverse transcriptases.
abstract: We have recently shown that reverse transcriptases (RTs) perform template switches when there is a very short (two-nucleotide) complementarity between the 3' ends of the primer (donor) strand and the DNA or RNA template acceptor strands [Oz-Gleenberg et al. (2011) Nucleic Acids Res 39, 1042-1053]. These dinucleotide pairs are stabilized by RTs that are capable of 'clamping' together the otherwise unstable duplexes. This RT-driven stabilization of the micro-homology sequence promotes efficient DNA synthesis. In the present study, we have examined several factors associated with the sequence and structure of the DNA substrate that are critical for the clamp activity of RTs from human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV), bovine immunodeficiency virus (BIV) and the long terminal repeat retrotransposon Tf1. The parameters studied were the minimal complementarity length between the primer and functional template termini that sustains stable clamps, the effects of gaps between the two template strands on the clamp activity of the tested RTs, the effects of template end phosphorylations on the RT-associated clamp activities, and clamp activity with a long 'hairpin' double-stranded primer comprising both the primer and the complementary non-functional template strands. The results show that the substrate conditions for clamp activity of HIV-1 and MLV RTs are more stringent, while Tf1 and BIV RTs show clamp activity under less rigorous substrate conditions. These differences shed light on the dissimilarities in catalytic activities of RTs, and suggest that clamp activity may be a potential new target for anti-retroviral drugs.
authors.affiliation: Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Iris
authors.initials: I
authors.lastname: Oz-Gleenberg
authors.forename: Eytan
authors.initials: E
authors.lastname: Herzig
authors.forename: Nickolay
authors.initials: N
authors.lastname: Voronin
authors.forename: Amnon
authors.initials: A
authors.lastname: Hizi
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016087:Immunodeficiency Virus, Bovine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D018626:Retroelements; D013379:Substrate Specificity; D013698:Templates, Genetic
chemical_list: D004279:DNA, Viral; D018626:Retroelements; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase