Proc. Natl. Acad. Sci. USA 97, 12451-12456
Polymerization
of non-template bases prior to transcription initiation at the 3 ends
of templates by an RNA-dependent RNA polymerase:
An activity involved in 3-end repair of viral RNAs
Hancheng Guan and Anne E. Simon
Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003
The 3 ends of RNAs associated with turnip crinkle virus (TCV), including subviral satellite satC, terminate with the motif CCUGCCC-3. Transcripts of satC with a deletion of the motif are repaired to wild-type (wt) in vivo by RNA-dependent RNA polymerase (RdRp)-mediated extension of abortively synthesized oligoribonucleotide primers complementary to the 3 end of the TCV genomic RNA (Nagy PD, Carpenter CD, Simon AE, 1997, Proc. Natl. Acad. Sci. USA, 94:1113-1118). Repair of shorter deletions, however, are repaired by other mechanisms. SatC transcripts with the 3-terminal CCC replaced by 8 non-viral bases were repaired in plants by homologous recombination between the similar 3 ends of satC and TCV. Transcripts with deletions of 4 or 5 3-terminal bases, in the presence or absence of non-viral bases, generated progeny with a mixture of wt and non-wt 3 ends. RdRp-containing extracts were able to polymerize nucleotides in a template-independent fashion before using these primers to initiate transcription at or near the 3 end of truncated satC templates. The non-template additions at the 5 ends of the nascent complementary strands were not random, with a preference for consecutive identical nucleotides. The RdRp was also able to initiate transcription opposite cytidylate, uridylate, guanylate and possibly adenylate residues without exhibiting an obvious preference, flexibility previously unreported for viral RdRp. The unexpected existence of 3 different repair mechanisms for TCV suggests that 3end reconstruction is critical to virus survival.