BISULFITE INDUCES TANDEM DOUBLE CC-]TT MUTATIONS IN DOUBLE-STRANDED DNA .2. KINETICS OF CYTOSINE DEAMINATION

Deamination of cytosine to uracil in double-stranded DNA (ds DNA) by sodium bisulfite has been monitored with a sensitive genetic assay. In this system, reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 C141 was detected by employing an ung bacterial strain defective in the enzyme uracil glycosylase. Within the 4-base target, it is possible to measure the rates of induction of C --> T, C --> A, C --> G, and CC --> TT mutations in DNA that has been incubated at physiological temperature and pH and then transfected into ung+ and ung- E. coli cells, respectively, for amplification and detection of the mutation. For concentrations of bisulfite from 1 to 50 mM, the reversion frequency in ung- cells increased linearly with time of incubation. The most interesting features of the bisulfite reaction were as follow: (1) Mutations were reduced 5-fold in ung+ cells, indicating ung is involved in repair of bisulfite-treated transforming DNA. (2) Sequencing of 157 revertants revealed that C --> T and tandem CC --> TT transition mutations comprised 100% of the mutations scored. (3) A unique finding was that, at the highest concentrations and longest incubation times, almost every mutant obtained in ds DNA exposed to bisulfite was found to be a CC --> TT tandem double mutation. (4) The high frequency of tandem double mutants is inconsistent with two random, independent mutational events and, coupled with the observed ung dependence, lends support to the concept of catalytic deamination, wherein bisulfite induces deamination in contiguous cytosines by a concerted mechanism. (5) Equations were derived for calculating the rate constant per site based on the data from DNA sequencing. Mutational rate constants at four cytosines in the ds DNA target at 37-degrees-C in 10 mM sodium bisulfite, 10 mM Hepes, pH 7.4 varied from 0.06 x 10(-10) s-1 to 0.35 x 10(-10) s-1 and showed a sequence context effect. A cytosine bordered on both sides by cytosine residues exhibited a mutation rate which was twice as great as a cytosine having only one nearest-neighbor cytosine. (6) In a tandem double CC --> TT mutation, there was a 4-5 order of magnitude increase in mutational rate constant of the second genetic event. These findings show that tandem double mutations, which have traditionally been ascribed to UV damage, can also be caused by chemical damage.