PubMed · 8569496
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M W Germann, N Zhou, J H van de Sande, H J Vogel. 1995. Parallel-stranded duplex DNA: an NMR perspective.. https://doi.org/10.1016/s0076-6879(95)61011-1
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Mechanical inoculation of Nicotiana tabacum with the PSTVd isolate KF 440-2 from the host plant tomato resulted in the de novo emergence, replication, and accumulation of a new "tobacco variant," designated PSTVd NT. It produces no symptoms in tobacco but, like PSTV KF 440-2, severe ones in tomato. The sequence analysis of PSTVd NT revealed a single nucleotide substitution from C-->U at position 259. Autonomous viroid replication was also induced in tobacco by genomic integration of oligomeric cDNA copies of PSTVd KF 440-2. Although these cDNAs contained the original tomato-specific C259, the circular PSTVd RNA subsequently accumulating in tobacco also exhibited the C259-->U259 substitution. In the secondary structure of PSTVd, nucleotide 259 is part of an internal loop analogous to loop E of eukaryotic 5S rRNA and presumed to be the only bulged extrahelical nucleotide of this loop. The C259 in PSTVd KF 440-2 and in practically all other isolates and the U259 in PSTVd NT of the loop E-like structure might be involved in protein binding and in viroid processing. The new variant PSTVd NT is genetically stable in both tobacco and tomato.
Recently, Draper and co-workers solved the structure of a hexanucleotide hairpin loop that is conserved in large subunit ribosomal RNAs. (In Escherichia coli, the hexanucleotide consists of nucleotides 1093 to 1098, in the GTPase center of 23 S rRNA.) A major feature of that structure is a G1093xA1098 base-pair that closes the loop. Our laboratory reported previously the isolation of the mutation G1093A and its characterization as a suppressor of UGA mutations and a cause of temperature-conditional lethality. For the work reported here, we asked whether G1093A causes its phenotypes precisely because it is part of the G1093/A1098 base-pair. Using oligonucleotide-directed site-specific mutagenesis, we introduced base substitutions at nucleotides 1093 and 1098 into a plasmid-borne ribosomal RNA operon (rrnB). Each mutant plasmid was then tested for the two mutant phenotypes, nonsense suppression and temperature-dependent growth inhibition. Our results indicate that mutations at 1093 do not cause the mutant phenotypes because G1093 is part of the G1093xA1098 base-pair. We discuss alternative avenues to the observed mutant phenotypes and, in particular, present a model in which a specific interaction of the loop is involved in peptide chain termination.
O6-Alkylguanine-DNA alkyltransferase (AGT) is an important cellular defense against the mutagenic effects of DNA alkylating agents. In humans this defense can contribute to the ability of some tumors to resist the effects of chemotherapeutic agents that act through DNA alkylation. We report here studies that characterize the interaction of AGT with DNA. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to single stranded deoxyribonucleotides. The stoichiometries of complexes formed with 16-, 30-, and 80-base oligodeoxyribonucleotides are 3.8 +/- 0.3, 5.3 +/- 0.2, and 8.9 +/- 0.2, respectively; the binding density decreasing from approximately 4 nt/monomer to approximately 9 nt/monomer as DNA length increases over this range. Binding competition assays show that DNA affinities depend only weakly on base composition or secondary structure, although in general G + C-rich sequences are bound with greater affinity than are A + T-rich ones and single-stranded DNA is bound with greater affinity than duplex forms. These results suggest mechanisms by which AGT may search for alkylated sites and interact with them to effect DNA repair.