2-Hydroxyadenine (A?) is formed from adenine during reaction of DNA with oxygen radicals. The enol form of A* pairs with T whereas the keto form with 5-methylisocytosine (iCM). We studied the influence of neighbouring bases in template, temperature and kind of polymerase on base-pairing of A" by comparison of relative efficiency of incorporation of d'ITP and diCMTP (calculated as the ratio of appropriate Vmax/Km) opposite A" site specifically located in oligodeoxynucleotide templates. We have revealed that the base pairing of A* is significantly influenced by template neighbours but not by temperature (3740°C) when studied in taq polymerase system.
A major pathway of mutagenesis in Escherichia coli is mediated by the inducible SOS response. Current models of SOS mutagenesis invoke the interaction of RecA and UmuD 2C proteins with a stalled DNA replication complex at sites of DNA lesions or poorly extendable terminal mismatches, resulting in an (error-prone) continuation of DNA synthesis. The precise mechanisms of SOS-mediated lesion bypass or mismatch extension are not known. Here, we have studied mutagenesis on the E. coli chromosome in recA730 strains. In recA730 strains, the SOS system is expressed constitutively, resulting in a spontaneous mutator effect (SOS mutator) because of reduced replication fidelity. We investigated whether during SOS mutator activity replication fidelity might be altered differentially in the leading and lagging strand of replication. Pairs of recA730 strains were constructed differing in the orientation of the lac operon relative to the origin of replication. The strains were also mismatch-repair defective (mutL) to facilitate scoring of replication errors. Within each pair, a given lac sequence is replicated by the leading-strand machinery in one orientation and by the laggingstrand machinery in the other orientation. Measurements of defined lac mutant frequencies in such pairs revealed large differences between the two orientations. Furthermore, in all cases, the frequency bias was the opposite of that seen in normal cells. We suggest that, for the lacZ target used in this study, SOS mutator activity operates with very different efficiency in the two strands. Specifically, the lagging strand of replication appears most susceptible to the SOS mutator effect.
We have investigated whether UV-induced mutations are created with equal efficiency on the leading and lagging strands of DNA replication. We employed an assay system that permits measurement of mutagenesis in the lacZ gene in pairs of near-identical strains. Within each pair, the strains differ only in the orientation of the lacZ gene with respect to the origin of DNA replication. Depending on this orientation, any lacZ target sequence will be replicated in one orientation as a leading strand and as a lagging strand in the other orientation. In contrast to previous results obtained for mutations resulting from spontaneous replication errors or mutations resulting from the spontaneous SOS mutator effect, measurements of UV-induced mutagenesis in uvrA strains fail to show significant differences between the two target orientations. These data suggest that SOS-mediated mutagenic translesion synthesis on the Escherichia coli chromosome may occur with equal or similar probability on leading and lagging strands.Exposure of cells to UV light results in the formation of mutagenic DNA lesions, among which the two most frequent lesions are cyclobutane pyrimidine dimers and 6-4 pyrimidinepyrimidone photoproducts at adjacent pyrimidines (13). In the bacterium Escherichia coli, mutagenesis by UV light and by a variety of chemical mutagens is mediated by the inducible SOS response (13,29). The SOS response is a global response involving the coordinated expression of some 30 genes (9). Its general function is to promote cellular survival upon induction of DNA damage, albeit at the cost of increased mutagenesis. The response is controlled by the interplay of the RecA and LexA proteins, the latter constituting the transcriptional repressor of the SOS regulon (13). After blockage of ongoing DNA replication by DNA damage, RecA forms nucleoprotein filaments with the damage-induced single-stranded DNA. This activated form of RecA then mediates the cleavage of LexA, thereby enabling the expression of the SOS genes.SOS mutagenesis is dependent on the lexA-controlled umuDC operon (17,18,32), which upon induction of the system yields the UmuDЈ 2 C complex (UmuDЈ being the RecAmediated cleavage product of UmuD). This complex was recently shown to be a DNA polymerase, termed Pol V (26,28,(34)(35)(36). Pol V is thought to promote mutagenesis by mediating mutagenic bypass of the DNA lesions, such as pyrimidine dimers, after DNA Pol III holoenzyme (HE) is blocked at such lesions. Pol V is a member of a recently described class of DNA polymerases (the UmuC/DinB/Rad30/Rev1 superfamily), now named Y-family DNA polymerases, that is found in a broad range of organisms (25). These enzymes are best characterized by their generally low-fidelity synthesis and/or ability to bypass DNA lesions in vitro (for a review, see reference 38). In addition to Pol V, E. coli also possesses another SOSinducible Y-family DNA polymerase, Pol IV (37). The precise functions of Pol IV are not yet clear, but its role in DNAdamage induced mutagenesis, including UV muta...
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