Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis

Abstract

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.

Replication of cyclobutane pyrimidine dimer analogue by Ex Taq DNA polymerase

“…While non-enzymatic ligation methods may offer some advantages, one limitation of the photoligation strategy relative to enzymatic methods is the fact that the CPD analogue as the ligated structures differs from that of the natural DNA junction, which is likely to interfere with further manipulation (such as amplification). However, in vitro analyses have shown that some DNA polymerases can copy distorted DNA templates containing a UV-induced lesion [16][17][18]. Additionally, because a single bond is included in the structure, d(T-CV U) has higher flexibility than usual CPD (Fig.…”

Replicative DNA Polymerase

“…Although Reuven et al, who have successfully reconstituted UmuDЈ 2 Cdependent lesion bypass in vitro, found it to be completely independent of the ␤ clamp of Pol III (43), Tang et al, using a slightly different approach to reconstitute UmuDЈ 2 C-dependent TLS in vitro (63), reported a strict requirement of both the ␤ clamp and the five-subunit clamp loader (␥ complex) of Pol III for lesion bypass. This difference with respect to the requirement of the ␤ clamp for TLS in vitro has been attributed to differences in the template DNAs and the levels of RecA protein used by these two groups (62,67), although it could also be due to the maltose binding protein-UmuC fusion protein utilized by Reuven et al (67).…”

“…Alternatively, it is possible that interactions of ␤ with the umuDC gene products are important for both the DNA damage checkpoint control (57) and TLS (62,63) and that self-cleavage of UmuD to yield UmuDЈ serves to modulate the interactions of UmuD 2 C and UmuDЈ 2 C with ␤ to effect the release of the checkpoint control and enable TLS (57). In any event, a definitive answer regarding the role of the ␤ clamp in TLS in the living cell will likely require a combined biochemical and genetic approach.…”

“…The umuDC genes encode a DNA polymerase, DNA polymerase V (Pol V), that has a remarkable ability to copy over abasic sites (43,63), cyclobutane dimers (62), and pyrimidinepyrimidone photoproducts (62), a process referred to as translesion DNA synthesis (TLS). This ability, however, comes at the cost of reduced fidelity.…”

“…This ability, however, comes at the cost of reduced fidelity. Thus, replication by Pol V is inherently less accurate, leading to the formation of mutations, even when replicating undamaged templates (29,62). Therefore, to limit the ability of Pol V to introduce mutations into the host genome, expression of the umuDC genes is tightly regulated as part of the Escherichia coli stress-induced SOS response (12).…”