| SOS mutagenesis requiring the UmuD'C proteins occurs as part of the cells' global response to DNA damage. The biochemical mechanism of SOS mutagenesis had remained a mystery due to the difficulties in obtaining biologically active UmuC protein, which is insoluble in aqueous solution when overproduced. Using purified UmuD'2C complex in its native form, we have reconstituted an SOS lesion bypass system in vitro.;We discovered that UmuD'2C is the fifth DNA polymerase in E coli. Efficient lesion bypass requires the function of pol V mutasome, consisting of pol V, activated RecA protein (RecA*), single-stranded binding protein (SSB), processivity factor beta sliding clamp, and gamma clamp loading complex. The effect of RecA on pol V causes a dramatic increase in DNA synthesis efficiency. Pol V is nonprocessive and competes with replicative pol III at lesion sites for free primer-3'-OH end. Yet it can interact with pol III in the vicinity of lesion sites, allowing pol III to take over after lesion bypass occurs.;We have examined the efficiency and specificity of pol V in bypassing common template lesions: pyrimidine-pyrimidone (6-4) photoproduct, cis-syn cyclobutane dimer, and abasic site. Pol V bypasses all three lesions within 30 seconds, whereas there is no detectable bypass by either pol III or pol IV on this time scale. A mutagenic "signature" for pol V is its incorporation of G opposite the 3'-T of a TT (6-4) photoproduct, in agreement with mutational spectra. In contrast, pol III and pol IV incorporate A almost exclusively, albeit with greatly reduced efficiency.;We have also measured the nucleotide incorporation fidelity of pol V at undamaged DNA template sites, pol V exhibits low fidelity with misincorporation frequencies ranging from ∼10-3 to 10-4 ; pol IV is 5- to 10-fold more accurate. Both pol V and pol IV exhibit enhanced ability to extend mismatched primer ends. While a definitive role of pol IV in untargeted mutagenesis remains to be clarified, our results suggest that pol V is responsible for both targeted and untargeted SOS mutagenesis in vivo. |
