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.

Human DNA polymerase iota (pol(iota)) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that poliota is able to facilitate limited translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that pol(iota) misinserts T or G opposite the 3' T of the CPD approximately 1.5 times more frequently than the correct base, A. While pol(iota) is unable to extend the T.T mispair significantly, the G.T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A. T base pair. By comparison, pol(iota) readily misinserts two bases opposite a 6-4 thymine-thymine pyrimidine-pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that poliota possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

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.