The antiretroviral agents azidothymidine, stavudine, and didanosine have the identical mutational fingerprint in the rpoB region of Escherichia coli.

We looked at the mutational fingerprints of three antiretroviral (anti-HIV) agents, azidothymidine (AZT), stavudine (STAV), and didanosine (DIDA) in the rpoB system of Escherichia coli and compared them with each other and with the fingerprints of trimethoprim and of spontaneous mutations in a wild-type and a mutT background. All three agents gave virtually identical fingerprints in the wild-type background, causing only A:T→C:G changes at 3 of the 12 A:T→C:G possible sites among the total of 92 possible base substitution mutations, even though AZT and STAV are thymidine analogs but DIDA is an adenosine analog. As all three agents are reverse transcriptase inhibitors, and act as chain blockers, the common fingerprint may be a property of chain blocking agents.

Enhanced characterization of the thyA system for mutational analysis in Escherichia coli: Defining mutationally "hot" regions of the gene.

We have extensively characterized base substitution mutations in the 795 base pair (bp) long E. coli thyA gene to define as many of the base substitution mutational sites that inactivate the gene as possible. The resulting catalog of mutational sites constitutes a system with up to 5 times as many sites for monitoring each of the six base substitution mutations as the widely used rpoB/Rifr system. We have defined 75 sites for the G:C -> A:T transition, 68 sites for the G:C -> T:A transversion, 53 sites for the G:C -> C:G transversion, 49 sites for the A:T -> G:C transition, 39 sites for the A:T -> T:A transversion, and 59 sites for the A:T -> C:G transversion. The system is thus comprised of 343 base substitution mutations at 232 different base pairs, all of which can be sequenced with a single primer pair. This allows for the examination of mutational spectra using a more detailed probe of known mutations, while still allowing one to compare the number of repeated occurrences at specific sites. We have examined several mutagens and mutators with this system, and show its utility by looking at the spectrum of cisplatin, that has a single hotspot, underscoring the value of having as large an array of sites as possible at which one can monitor repeat occurrences. To test for regions of the gene that might be hotspots for a number of mutagens, or "hot" (mutaphilic) regions, we have looked at the ratio of mutations per set of an equal number of mutational sites throughout the gene. The resulting graphs suggest that there are "hot" regions at intervals, and this may reflect aspects of secondary structures, of the higher order structure of the chromosome, or perhaps the nucleoid structure of the chromosome plus histone-like protein complexes.

Mutations induced by Bleomycin, 4-nitroquinoline-1-oxide, and hydrogen peroxide in the rpoB gene of Escherichia coli: Perspective on Mutational Hotspots.

We report the mutational spectra in a segment of the E. coli rpoB gene of bleomycin (BLEO), 4-nitroquinoline-1-oxide (NQO), and hydrogen peroxide (H2O2). We compare these spectra with those of other mutagens and repair deficient strains in the same rpoB system, and review the key elements determining mutational hotspots and outline the questions that remain unanswered. We consider three tiers of hotspots that derive from 1) the nature of the sequence change at a specific base, 2) the direct nearest neighbors and 3) some aspect of the larger sequence context or the local 3D-structure of segments of DNA. This latter tier can have a profound effect on mutation frequencies, even among sites with identical nearest neighbor sequences. BLEO is dependent on the SOS-induced translesion Pol V for mutagenesis, and has a dramatic hotspot at a single mutational site in rpoB. NQO is not dependent on any of the translesion polymerases, in contrast to findings with plasmids treated in vitro and transformed into E. coli. The rpoB system allows one to monitor both G:C -> A:T transitions and G:C -> T:A transversions at the same site in 11 cases, each site having the identical sequence context for each of the two mutations. The combined preference for G:C -> A:T transitions at these sites is 20-fold. Several of the favored sites for hydrogen peroxide mutagenesis are not seen in the spectra of BLEO and NQO mutations, indicating that mutagenesis from reactive oxygen species is not a major cause of BLEO or NQO mutagenesis, but rather specific adducts. The variance in mutation rates at sites with identical nearest neighbors suggests that the local structure of different DNA segments is an important factor in mutational hotspots.