FUNCTIONAL RECA, LEXA, UMUD, UMUC, POLA, AND POLB GENES ARE NOT REQUIRED FOR THE ESCHERICHIA-COLI UVM RESPONSE

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N-4-ethenocytosine, epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in Delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in Delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in Delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence-of detectable induction of dinD, an SOS gene. These results make it unlikely-that UVM results from a recA-independent alternative induction pathway for SOS genes. Similarly, UVM is observed in polA (deficient in DNA polymerase I) and polB (deficient in DNA polymerase II) cells, suggesting that neither polymerase plays an indispensable role in UVM induction. Furthermore, our data Show that the UVM response is accompanied by enhanced survival (UVM reactivation) of M13 DNA bearing epsilon C. The observation of UVM reactivation makes simple repair-suppression models for UVM less attractive and increases the plausibility of mechanisms operating at the level of base insertion. We hypothesize that noncoding lesions fall into two categories. The so-called SOS-dependent (class 1) lesions require SOS functions at the extension (bypass) step, whereas class 2 noncoding lesions do not. It is proposed that UVM, a previously unrecognized damage-inducible response, modulates base insertion at noncoding lesions.