The expression of the Escherichia coli DNA polymerases pol V (UmuD'2C complex) and pol IV (DinB) increases in response to DNA damage. The induction of pol V is accompanied by a substantial increase in mutations targeted at DNA template lesions in a process called SOS-induced error-prone repair. Here we show that the common DNA template lesions, TT (6-4) photoproducts, TT cis-syn photodimers and abasic sites, are efficiently bypassed within 30 seconds by pol V in the presence of activated RecA protein (RecA*), single-stranded binding protein (SSB) and pol III's processivity beta,gamma-complex. There is no detectable bypass by either pol IV or pol III on this time scale. A mutagenic 'signature' for pol V is its incorporation of guanine opposite the 3'-thymine of a TT (6-4) photoproduct, in agreement with mutational spectra. In contrast, pol III and pol IV incorporate adenine almost exclusively. When copying undamaged DNA, pol V exhibits low fidelity with error rates of around 10(-3) to 10(-4), with pol IV being 5- to 10-fold more accurate. The effects of RecA protein on pol V, and beta,gamma-complex on pol IV, cause a 15,000- and 3,000-fold increase in DNA synthesis efficiency, respectively. However, both polymerases exhibit low processivity, adding 6 to 8 nucleotides before dissociating. Lesion bypass by pol V does not require beta,gamma-complex in the presence of non-hydrolysable ATPgammaS, indicating that an intact RecA filament may be required for translesion synthesis.

The damage-inducible UmuD and UmuC proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD 2 C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD 2 C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD 2 C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, ␤ sliding clamp, ␥ clamp loading complex, and E. coli single-stranded binding protein (SSB), pol V's polymerase activity is highly ''error prone'' at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100-to 150-fold higher than pol III HE. In accordance with the ''A-rule,'' dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD 2 C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3-OH template-primer ends. We show, however, that despite competition for primer-3-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.

Damage-induced SOS mutagenesis requiring the UmuDC proteins occurs as part of the cells' global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD 2 C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD 2 C, activated RecA protein (RecA*), ␤-sliding clamp, ␥-clamp loading complex, single-stranded binding protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD 2 C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD 2 C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.