Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.
The sfiA and sfiB mutations, originally isolated in thermoresistant ultraviolet-resistant revertants of a tif lon strain, also suppressed filamentation in tsl strains (mutated at the lexA locus). When deoxyribonucleic acid synthesis was arrested, however, sfi-independent filamentation occurred. Other SOS functions were not affected by sfiA and sfiB mutations; in particular, ultraviolet-induced repair and mutagenesis of bacterial deoxyribonucleic acid were normal, as was tsl-tif-induced synthesis of recA protein. Genetic studies (i) established the identity of map location of the sfiA and sulA loci, (ii) showed that the two sfiB mutations are recessive, and (iii) showed that of six independent sfiA mutations, three are recessive and three are dominant. One sfiB strain was shown to have a 6% growth disadvantage relative to a sfi+ or sfiA strain. It is proposed that the sfiA locus may define the structural gene of a hypothetical inducible SOS-associated division inhibitor.
3-Nitropyrrole (M) was introduced as a non-discriminating 'universal' base in nucleic acid duplexes by virtue of small size and a presumed tendency to stack but not hydrogen bond with canonical bases. However, the absence of thermally-induced hyperchromic changes by single-stranded deoxyoligomers in which M alternates with A or C residues shows that M does not stack strongly with A or C nearest neighbors. Yet, the insertion of a centrally located M opposite any canonical base in a duplex is sometimes even less destabilizing than that of some mismatches, and the variation in duplex stability is small. In triplexes, on the other hand, an M residue centrally located in the third strand reduces triplex stability drastically even when the X.Y target base pair is A.T or G. C in a homopurine. homopyrimidine segment. But, when the target duplex opposition is M-T and the third strand residue is T, the presence of M in the test triplet has little effect on triplex stability. Therefore, a lack of hydrogen bonding in an otherwise helix-compatible test triplet cannot be responsible for triplex destabilization when M is the third strand residue. Thus, M is non-discriminating and none-too-destabilizing in a duplex, but in a triplex it is extremely destabilizing when in the third strand.
The effect of glycerol on the specificity of DNA cleavage by the restriction.endonuclease BamHI has been examined. In addition to the canonical G 4 G-A-T-C-C site, BamHI cuts DNA at several sites that we have named noncanonical BamHI. 1 sites. The number ofBamHI. 1 sites in simian virus 40 and pBR322 was determined to be 13 for each DNA. Cutting sites determined by DNA sequence analysis include G
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