MOLECULAR-BASIS OF NITROGEN-MUSTARD EFFECTS ON TRANSCRIPTION PROCESSES - ROLE OF DEPURINATION

DNA was alkylated with nitrogen mustard (HN2) and the rate of release of the alkylpurines was quantitated by HPLC. The half life of depurina tion of the major product (7-alkylguanine) was 9.1 h at 37 degrees C. End-labelled DNA was used to show that depurination occured dominantly at 5'-GA, 5'-GG and 5'-GT sequences. Although extensive alkylation wa s observed at all 5'-GNC and 5'GNT sequences, no depurination was obse rved at these sites during a depurination time of 20 h at 37 degrees C . Since these sites are potential interstrand crosslinking sequences ( G-adduct-G and G-adduct-A, both spanning an intervening base pair), th is suggests that these regions have a greatly enhanced stability or th at simultaneous depurination of both ends of the crosslink is necessar y before these lesions are removed (with a predicted half-life of appr oximately 80 h at 37 degrees C). Depurination at the lac UV5 promoter impaired the association of Escherichia coli RNA polymerase with that promoter, while in the elongation phase two distinctly different seque nce-specific processes were apparent. At 5'-GNC and 5'-GNT sequences t ranscriptional blockages were maintained with increasing elongation ti me, whereas at monoadduct sites, the blockage decreased with elongatio n time (predominantly at 5'-GG and 5'-GC sequences), with an average h alf-life of approximately 10.7 h. Collectively, these results suggest that the observed read-through past monoadduct sites is due to depurin ation of the DNA at those sites. E.coli RNA polymerase is therefore ab le to transcribe efficiently past apurinic sites and presumably does s o by incorporating an incorrect base into the nascent RNA.