Early events in the synthesis of the multicopy single-stranded DNA-RNA branched copolymer of Myxococcus xanthus.
Myxobacteria and a variety of strains of Escherichia coli contain an unusual extrachromosomal element, a small single-stranded branched copolymer of DNA and RNA (msDNA). Interest in msDNA stems from the presence of a 2'-5' linkage between its DNA and RNA moieties and the possible involvement of reverse transcriptase in its synthesis. Two groups have proposed a model for the synthesis of msDNA that involves the following sequence of events: 1) synthesis of an RNA precursor; 2) addition of a dNTP in a 2'-5' linkage to the RNA precursor (branch priming); 3) synthesis by reverse transcriptase of complementary DNA on the primed RNA precursor; 4) concomitant processing of the RNA precursor by RNase H; and 5) further processing of the RNA precursor by RNase H; and 5) further processing of the branched polymer. The branch priming hypothesis (step 2) was originally based on pulse-chase experiments using a lengthy pulse of 30-min duration. Our experiments with shorter pulse durations demonstrate a variety of intermediate forms not predicted by this model. Specifically, we find that early in synthesis, intermediate msDNA forms appear that are apparently not branch-linked to corresponding RNA moieties. The concomitant RNase H hypothesis (step 4) was based in part on intermediate trapping experiments using dideoxy NTPs to interrupt msDNA synthesis. These experiments showed an apparent complementary relationship between DNA length and RNA length in the trapped forms. In contrast, our experiments show that DNA elongation and RNA processing follow different kinetics. These results suggest an alternate model for synthesis of msDNA in which a conventionally primed DNA moiety is joined with the RNA moiety in a branch ligation event taking place several minutes after the synthesis of both moieties.
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Cell and Molecular Biology Program, University of Texas, Dallas, Richardson 75083.
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D001420:Bacteria, Aerobic / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D012329:RNA, Bacterial; C058681:msDNA; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H