Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I.

Abstract

Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment. Modificationis included the bulky guanine-8-aminoflqorene adduct and a guanine oxidation product resembling the product of photosensitized 1)NA oxidation. By a combination of primers and "nick-mers" conditions of single-strand-directed DNA synthesis and nick-translation could be created. Our results show that the polymerase can bypass both types of lesions. Bypass occurs on a single-stranded template but is facilitated on a nicked, double-stranded template. Only purines, with guanine more favored than adenine, are incorporated across both lesions. Hesitation during bypass coild not be detected. The results indicate that site-specifically modified oligonucleotides can be sensitive probes for the action of polymerases on damaged templates. They also suggest a function for polymerase I, in its nick-translation capacity, during DNA repair and mutagenesis.Mutation induced by gene damage is partially caused by the cell's polymerases. It is known that in Escherichia coli this involves a damage-induced or SOS response of great complexity, which allows the cell to overcome otherwise lethal damage at the expense of increased mutations (1, 2). Mutants of E. coli (umuC mutants) have been isolated in which error-prone repair is specifically affected (3). It has been suggested that the umuC gene product, part of the damageinduced response, is involved in a process of DNA replication that is inducible and able to synthesize DNA on a damaged template (4). This process might involve a temporary alteration of polymerase III (5). Another enzyme that appears to be involved in the damage-induced response is polymerase 1 (6). This enzyme is not inducible, but its suggested role in gap-filling, following excision repair and during postreplication repair, would imply that its nicked and gapped substrates would be available mostly under conditions of the induced SOS response.To improve our understanding of how polymerases respond to damaged templates, site-specifically modified oligonucleotides, in which a known position is substituted by a defined bulky carcinogen adduct, have been developed in this laboratory (7). Here we describe experiments designed to probe both the ability of polymerase I to replicate past a site-specifically modified template and the identity of the nucleotides inserted across from the modified base. MATERIALS AND METHODSSynthesis of Operators with Site-Specific Modifications. The synthesis of a 14-mer operator fragment modified with 8-aminofluorene has been described (7). For the present study, we have synthesized 17-mer, 18-mer, and 23-mer operator strands. The conventional identification of the transcribed strand as the top strand is used (8). Sequences 3' and 5' of the central 17-mer operator are restriction sites. Both the adoption of the phosphoramidite chemistry (9) and the purification by tritylselection (10) gave a much cleaner product as ju...

Effects of Aminofluorene and Acetylaminofluorene DNA Adducts on Transcriptional Elongation by RNA Polymerase II

“…2). Similar results have been reported for the effects of these lesions on transcription by T7 RNA polymerase (22) and on replication by DNA polymerases (18)(19)(20)(21). The translesional bypass of an AF adduct by RNA pol II we observed contrast with results reported for X. laevis RNA pol III, which was blocked by the presence of an AF adduct (23).…”

“…An AAF adduct is a much stronger block than an AF adduct to the progression of E. coli DNA polymerase I (18,19), T7 DNA polymerase (19)(20)(21), and T7 RNA polymerase (22). Both adducts, however, are strong blocks to transcription by RNA pol III from Xenopus laevis (23).…”

Site-specifically modified oligodeoxynucleotides as probes for the structural and biological effects of DNA-damaging agents

“…Obviously, the conclusion that an adduct is mutagenic would be buttressed if the biological results on it prove to be independent of the route of modified oligonucleotide preparation. As a second point, we and some of our colleagues (11)(12)(13)(14)(15) have emphasized the use of short oligonucleotides (4-to 7-mers), which are much easier to purify than the 14-to 20-mers used by others (16,17). It is also easier to assess the levels of contamination of short oligonucleotides since minor differences in composition usually cause substantial differences in mobility characteristics by conventional separation tools; these differences decrease in magnitude as the length of the oligonucleotide increases.…”

Mutation induced in vitro on a C-8 guanine aminofluorene containing template by a modified T7 DNA polymerase

“…Such bypass is hardly surprising in view of the published data indicating that multiple AF lesions are necessary to inactivate 0X174 DNA (Lutgerink et al, 1985) as well as biochemical evidence that these lesions are bypassed (Michaels et al, 1986;O'Connor & Stohrer, 1985).…”