Intrinsic Polymerase Activities of UmuD′ ₂ C and MucA′ ₂ B Are Responsible for Their Different Mutagenic Properties during Bypass of a T-T cis-syn Cyclobutane Dimer

Abstract

In wild-type Escherichia coli, translesion replication is largely dependent upon the UmuD 2 C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA 2 B. Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu-and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ ( subunit) or holE ( subunit) or carrying the dnaQ allele mutD5, which is deficient in proofreading but is competent in the structural function of , or the dnaE antimutator allele spq-2. The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuDC, MucAB, or MucAB proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu-and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA 2 B complex is more efficient in promoting translesion replication than the UmuD 2 C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA 2 B than by UmuD 2 C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass.It has been inferred, on the basis of a variety of genetic evidence obtained over the last 20 years, that translesion replication and DNA damage-induced mutagenesis (SOS mutagenesis) in Escherichia coli are dependent on the activity of DNA polymerase III (polIII) holoenzyme or a modified form of it (4,5,14,26,27; reviewed in reference 10). Although polIII is usually unable to perform lesion bypass by itself, it can do so with the aid of what were previously thought to be accessory factors, the chromosomally encoded UmuDC proteins or their plasmid-encoded homologs, such as MucAB (16,22,30). However, the recent discovery that the UmuDЈ 2 C protein complex, now called polV, itself possesses intrinsic DNA polymerase activity (24,25,32,33) raises the question of which enzyme is responsible for mutagenesis. Is polV solely responsible, or do some or all of the polIII subunits also influence SOS mutagenesis in vivo? In vitro studies show that polV is capable of both nucleotide incorporation opposite an abasic site and extension from this terminus in the absence of the polIII core (composed of the catalytic ␣ subunit, the 3Ј-5Ј exonucleolytic proofread...

Escherichia coliDNA Polymerase III Can Replicate Efficiently past a T-Tcis-synCyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded bydnaQAre Inactivated

“…In the UV-irradiated Pol Vproficient strains, TK603 and DV12, the mutations observed are predominantly T3A, with a few T3C (combined data show 45 T3A and 4 T3C). This result is in keeping with a large body of data from previous investigations (19,23,31). In strains lacking Pol V, however, no T3A mutations were observed (combined data, 0 T3A and 466 T3C).…”

“…Although at first sight it might seem that the greater laxity of Pol V in base selection is required for dimer bypass, this conclusion may well be wrong, since the distantly related human Pol also preferentially misincorporates T opposite the 3Ј T of the cis-syn T-T dimer yet bypasses the lesion inefficiently (34). Conversely, MucAЈB, a more closely related member of the Y family of DNA polymerases (24), bypasses the cis-syn T-T dimer more efficiently than Pol V, yet resembles Pol III in its misincorporation specificity; like Pol III, it has a twofold-lower error rate than Pol V and generates more than sevenfold more T3C than T3A mutations (23). Thus, it seems more likely that the efficiency of lesion bypass and the differences one observes in mutational spectra between various members of the Y family of DNA polymerases arise from just a few amino acid substitutions at key locations within the active site of each enzyme.…”

The aflatoxin B 1 formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma

“…Phage DNA harboring the AFB 1 -FAPY adduct survives best when replicated in cells expressing MucAB, a polymerase that is currently thought to be the most proficient at bypass of bulky lesions (Fig. 2B) (17,50,(52)(53)(54)(55)(56)(57)(58). This study has established that the FAPY major adduct is the strongest block to replication of all of the aflatoxin adducts studied here, irrespective of which bypass polymerase is used by the cell to help it overcome the DNA damage.…”

The role of conjugative transposons in the Enterobacteriaceae

“…UmuDC has been shown to encode a new type of polymerase termed DNA polymerase V that polymerizes in an error-prone manner past DNA damage without template assistance [30]. The CTnR391 RumAB proteins have also been shown to possess DNA polymerase V activity, although with different specificity of error-prone base insertion than the chromosomal homologue [31]. The ability of mobile elements to enhance mutagenesis and repair in stressed environments obviously has evolutionary and survival advantages and is quite widely distributed amongst mobile elements.…”