Mechanism of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine.

Abstract

We measured the kinetics of growth and mutagenesis of UV-irradiated DNA of phages S13 and A that were undergoing SOS repair; the kinetics strongly suggest that most of SOS mutagenesis arises from the deamination of cytosine in cyclobutane pyrimidine dimers, producing C -3 T transitions. This occurs because the SOS mechanism bypasses T'T dimers promptly, while bypass of cytosine-containing dimers is delayed long enough for deamination to occur. The mutations are thus primarily the product of a faithful mechanism of lesion bypass by a DNA polymerase and are not, as had been generally thought, the product of an error-prone mechanism. All of these observations are explained by the A-rule, which is that adenine nucleotides are inserted noninstructionally opposite DNA lesions.SOS repair and SOS mutagenesis were discovered nearly 40 years ago by Jean Weigle, who observed that preirradiation of the bacterial host increased survival of UV-irradiated phage A (1). When applied to phage DNA these SOS phenomena are now called Weigle reactivation and Weigle mutagenesis; their mechanism has thus been an intriguing puzzle for some time. We show here that the primary, but not the sole, mechanism of UV mutagenesis in phages S13 and A is simply the spontaneous deamination of cytosine in cyclobutane pyrimidine dimers. This deamination occurs during a deliberate, and arguably an unnecessary, delay in the bypass of the lesion by DNA polymerase; the delay is caused by cytosine-containing dimers, possibly because of the mispairing of the cytosine with adenine. SOS repair is induced by damage to DNA. In Escherichia coli, the damage results in an activated form of the RecA protein, which can mediate the cleavage of LexA, the repressor ofthe SOS regulon. The principal known components of the repair system are the products of the recA and the umuDC genes. The activated RecA protein is needed for cleavage of the UmuD protein to produce UmuD', the active C-terminal fragment (2-4). The RecA protein can also be activated by mutations designated recA(Prtc) to what is termed the protease constitutive state (5); with mutants such as recAl202(Prtc) and recAJ237(Prtc), which were used in the work described here, Weigle reactivation is achieved without irradiation of the host cell.It is generally thought that a high frequency of erroneous base pairing during DNA synthesis opposite the distorted lesion is responsible for the mutagenesis that accompanies SOS repair, and that idea has evoked the term error-prone repair. We propose, instead, that most, though certainly not all, ofthe mutations arise through accurate base pairing-i.e., an error-free bypass mechanism. This paradox will be resolved by the fact that mutations can arise by deamination of cytosine in cyclobutane dimers; if the uracils formed are read correctly during bypass of the dimers, adenines would be found in the newly synthesized complementary strand. If there is adequate time for deamination to occur, and that is the very central issue here, bypass of lesions will usually invo...

Requirement of DNA Polymerase η for Error-Free Bypass of UV-Induced CC and TC Photoproducts

“…In Escherichia coli, such deamination has been proposed to account for the C3T transitions which then occur because of accurate insertion of adenines opposite uracils resulting from cytosine deamination (26). If Pol were to replicate through uracil-containing dimers by inserting an A opposite a U, as it does so efficiently for the TT dimer, in that case, inactivation of Pol should reduce the frequency of UV-induced mutations at the TC and CC sites.…”

Mutagenic properties of the T-C cyclobutane dimer

“…This model is feasible only if the deamination rate is high, and on this issue there is conflicting evidence. Although Tessman and colleagues provided indirect evidence that the half-life (t 1/2 ) for deamination is 30 to 60 min, other evidence suggests that it is likely to be 2 to 12 h (3,6,8,9,19,22,(24)(25)(26), a rate that is probably too low for deamination to be a significant feature of mutagenesis in Escherichia coli, though it might be significant in organisms with longer cell cycles.An alternative model to explain the high incidence of UVinduced mutations at T-C and C-C sites might be that the cytosine-containing dimers, in contrast to the T-T (1), T-U (13), and U-U (10) photoproducts, are intrinsically highly mutagenic because of either a high error rate during translesion replication, a high rate of bypass, or a combination of these factors. We investigated the reasons for the high mutability of the cytosine-containing photoproducts by constructing vectors carrying a specifically located T-C dimer, followed by transfection into SOS-induced and uninduced E. coli cells and sequence analysis of the replicated products.…”

“…This model is feasible only if the deamination rate is high, and on this issue there is conflicting evidence. Although Tessman and colleagues provided indirect evidence that the half-life (t 1/2 ) for deamination is 30 to 60 min, other evidence suggests that it is likely to be 2 to 12 h (3,6,8,9,19,22,(24)(25)(26), a rate that is probably too low for deamination to be a significant feature of mutagenesis in Escherichia coli, though it might be significant in organisms with longer cell cycles.…”

“…One explanation invokes the high rate of deamination of cytosine to uracil within dimers (8,9,21,22,(24)(25)(26), which results in G ⅐ C3A ⅐ T transitions, usually the most abundant substitution at these sites. In a recent investigation and elaboration of this model, Tessman and colleagues proposed additionally that the cytosine dimers present a much more potent block to replication than their thymine-or uracil-containing counterparts and that dimers are generally bypassed only after deamination has occurred (25,26), thus guaranteeing a highly mutagenic outcome. This model is feasible only if the deamination rate is high, and on this issue there is conflicting evidence.…”

DNA repair mutants of Rhodobacter sphaeroides

“…Being photosynthetic, it is subjected to potentially lethal UV irradiation, making its mode of DNA repair of special interest. The high GϩC content (65%) of the R. sphaeroides genome makes this of further interest because of the expected higher content of Z DNA, the greater need for uracil repair following cytosine deamination, and the role of the latter process in UV mutagenesis when it occurs in pyrimidine dimers (49). In addition, R. sphaeroides can use a range of compounds as terminal electron acceptors, including toxic metal oxides and organic compounds with a potential for free radical formation (34).…”