A proflavin-induced frameshift hotspot in the thymidylate synthase gene of bacteriophage T4.
Twenty-one independent thymidylate synthase deficient (td) mutants were isolated after proflavin mutagenesis of T4D0 phage. A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5' splice site of this intron-containing gene. This region comprises only 14% of the total td exon sequence. RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3' end of exon I of the td gene. The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3' ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine. Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phosphodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA. Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis. The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4.
Brown
M D
MD
School of Biology, Georgia Institute of Technology, Atlanta 30332-0230.
Ripley
L S
LS
Hall
D H
DH
D017122:Bacteriophage T4 / Q000235:genetics; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004279:DNA, Viral; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D009153:Mutagens / Q000633:toxicity*; D011370:Proflavine / Q000633:toxicity*; D015183:Restriction Mapping; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism
D004279:DNA, Viral; D009153:Mutagens; D011370:Proflavine; D013940:Thymidylate Synthase