Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase.
Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication.
Department of Microbe-Plant Interactions, CBIB (Center for Biomolecular Interactions Bremen), University of Bremen, PO Box 330440, D-28334 Bremen, Germany.
Jessica
J
Döring
Department of Microbe-Plant Interactions, CBIB (Center for Biomolecular Interactions Bremen), University of Bremen, PO Box 330440, D-28334 Bremen, Germany.
Thomas
T
Hurek
D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009154:Mutation; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000302:isolation & purification; D059367:RNA Cleavage; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D048348:Reverse Transcription; D016914:Ribonuclease H / Q000235:genetics; D013698:Templates, Genetic
D018076:DNA, Complementary; D009843:Oligoribonucleotides; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H