DNA strand-specific repair of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene adducts in the hamster dihydrofolate reductase gene.

Abstract

We evaluated the formation and removal of (+)-3a,48-dihydroxy-la,2a-epoxy-1,2,3,4-tetrahydroben- 2). A similar strand bias for BPDE-induced mutations in the hypoxanthine phosphoribosyl transferase gene (HPRT) of human fibroblasts was described where 77% of the premutagenic guanine bases were also on this strand (3). Assuming that carcinogen modification of DNA in vivo occurs with similar frequency on both strands, several possible explanations for the apparent strand selectivity of induced mutations can be considered and tested. One explanation for the strand bias of mutations is that the selection for dihydrofolate reductase-deficient mutants is very stringent, causing mutations to occur on one strand preferentially (4). We recently devised a reversion assay to address this possibility (5). BcPHDE-induced forward mutations were transversions favored at triple purine sequences where the 5' base was altered (2). The assay used DHFRnonsense mutants that carry this target sequence on both strands at each of two stop codons, and all purine thymine mutations yield amino acid substitutions compatible with dihydrofolate reductase activity. Randomly induced mutations at these sites would produce a 2:1 ratio of substitutions on the nontranscribed versus the transcribed strand. Sequence analysis of 66 independent BcPHDE-induced revertants showed that the combined ratio of mutations affecting purines on the nontranscribed strand was 15:1. Hence, the strand bias here was independent of phenotypic selection.Analysis of induced mutations in rodent HPRT led to the suggestion that strand-biased mutations relate to the polarity of the marker gene with respect to DNA replication (6). Since strand-biased UV-induced mutations were found in the absence of DNA repair on the transcribed strand of HPRT (7), the misinsertion rate differences for replicative polymerases a and 6 may also contribute to the strand-bias phenomenon.

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

“…EMS mainly induces GC to AT transitions (2), and BcPHDE induces purine-to-T transversions in mammalian cells (10). Perusal of the substrate sequences (Table 2) shows that if fixation of induced mutations occurs preferentially on the nontranscribed strand (5,12), this mutagen action could account for much of the specificity, since there are no G or A substitutions that would improve the splice donors at positions +3, +4, +5, and +6. Changes in the exonic positions -1, -2, and -3 are obviously constrained by the necessity to effect nondetrimental amino acid changes in the enzyme.…”

DNA repair defects associated with chromosomal translocation breaksite regions.

“…There is reason to believe that strand-independent repair mechanisms may also serve a biological function-to maintain genetic stability rather than to promote cellular survival. Recent studies in rodent cells have shown that selectively repaired genes (template strand restricted) can be readily mutagenized by an accumulation of premutagenic lesions in the poorly repaired, nontemplate strand (8,18,36 phoblasts demonstrates a consistent correlation between repair deficiency and translocation breaksite regions.…”

Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: relating biological consequences to conformational preferences

“…The effects of trans-anti-[BPh]-N 6 -dA adducts on RNA polymerase II elongation is of particular interest because these lesions are poor substrates for NER, leaving TCR as an important pathway for their removal (26,45). (+)-1R-and (±)-1S-trans-anti-[BPh]-N 6 -dA lesions induce relatively less perturbations to double-stranded DNA compared with the analogous adducts derived from benzo[a]pyrene (46); this fact, coupled with the absence of damaged duplex destabilization and optimized stacking of the stereoisomeric adducts with the DNA, explains why NER recognizes these lesions poorly when they are present in the genome (46,47).…”

“…Little is known about the behavior of human RNA polymerase II at BPhDE lesions in DNA. Considering that there is a strand bias in mutagenesis for BPhDE adducts in DNA (15) that may well result from TCR operating on these lesions (26), it was of interest to assess the behavior of RNA polymerase II during transcription elongation past BPhDE lesions in DNA.…”