Proc. Natl. Acad. Sci. USA Vol. 89, pp. 1159-1163, February 1992 Genetics Mechanism of SOS mutagenesis of UV-irradiated DNA: Mostly error-free processing of deaminated cytosine (A-rule/umusDC/groE/pyrimidine dimers/phages S13 and A) IRWIN TESSMAN, SHI-KAU Liu, AND MATTHEW A. KENNEDY Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 Communicated by R. B. Setlow, November 8, 1991 (receivedfor review July 16, 1991) ABSTRACT We measured the kinetics of growth and correctly during bypass of the dimers, adenines would be mutagenesis of UV-irradiated DNA of phages S13 and A that found in the newly synthesized complementary strand. If were undergoing SOS repair; the kinetics strongly suggest that there is adequate time for deamination to occur, and that is most of SOS mutagenesis arises from the deamination of the very central issue here, bypass of lesions will usually cytosine in cyclobutane pyrimidine dimers, producing C -3 T involve the insertion of the sequence A-A opposite cyclob- transitions. This occurs because the SOS mechanism bypasses utane dimers, even opposite those dimers that had originally T'T dimers promptly, while bypass of cytosine-containing contained C. Because of the distorted template, partially dimers is delayed long enough for deamination to occur. The relaxed proofreading might be needed to facilitate the accu- mutations are thus primarily the product of a faithful mech- rate bypass (6, 7). anism of lesion bypass by a DNA polymerase and are not, as The foundation for the current work lies in our study of had been generally thought, the product of an error-prone mutagenesis by delayed photoreactivation of UV-irradiated mechanism. All of these observations are explained by the phage S13 and its naked DNA (8). In that work we attributed A-rule, which is that adenine nucleotides are inserted nonin- all of the mutagenesis to the deamination of cytosine, which structionally opposite DNA lesions. occurs readily when the 5,6 bond of the base is saturated (9, 10), as it is in cyclobutane dimers. We proposed, therefore, SOS repair and SOS mutagenesis were discovered nearly 40 that most SOS mutagenesis of UV-irradiated DNA might Jean result from error-free translesion DNA synthesis after deam- years ago by Weigle, who observed that preirradiation ination occurs. The kinetics ofthe mutagenic effect produced of the bacterial host increased survival of UV-irradiated by delayed photoreactivation is an unusual step function: phage A (1). When applied to phage DNA these SOS phe- storage of UV-irradiated phage for 15 min at 37°C is not nomena are now called Weigle reactivation and Weigle mutagenic, indicating that deamination is negligible in that mutagenesis; their mechanism has thus been an intriguing time, but an additional 15 min of storage is highly mutagenic, puzzle for some time. We show here that the primary, but not suggesting that deamination of practically all available sites the sole, mechanism of UV mutagenesis in phages S13 and A occurs by 30 min (8). More detailed experiments with infec- is simply the spontaneous deamination of cytosine in cyclo- tious S13 DNA show that the transition from negligible to butane pyrimidine dimers. This deamination occurs during a nearly complete mutagenesis occurs between 25 and 30 min deliberate, and arguably an unnecessary, delay in the bypass ofDNA storage at 37°C (unpublished data). Earlier studies of of the lesion by DNA polymerase; the delay is caused by mutagenesis by delayed photoreactivation had already indi- cytosine-containing dimers, possibly because of the mispair- cated that dimerized cytosines in the double-stranded DNA ing of the cytosine with adenine. of E. coli are deaminated in vivo within 90 min at 37°C (11). SOS repair is induced by damage to DNA. In Escherichia We predicted that if a sufficiently long delay were an intrinsic coli, the damage results in an activated form of the RecA aspect of the SOS repair process, deamination of the cy- protein, which can mediate the cleavage of LexA, the re- tosines in UV-induced dimers could have time to occur and pressor ofthe SOS regulon. The principal known components would then be a source of the C -+ T mutations that are so of the repair system are the products of the recA and the commonly induced by UV irradiation. We show here that umuDC genes. The activated RecA protein is needed for Weigle mutagenesis is indeed characterized by a delay in cleavage of the UmuD protein to produce UmuD', the active repair that is on the order of the time needed for deamination C-terminal fragment (2-4). The RecA protein can also be of dimerized cytosines. activated by mutations designated recA(Prtc) to what is Weigle reactivation of phage S13 occurs effectively in a termed the protease constitutive state (5); with mutants such groE mutant, but 80% of the mutagenesis is eliminated (7). as recAl202(Prtc) and recAJ237(Prtc), which were used in the We will describe here a recA mutant that accomplishes work described here, Weigle reactivation is achieved without essentially the same thing, namely, efficient reactivation of irradiation of the host cell. S13, and of phage A too, but with severely reduced muta- It is generally thought that a high frequency of erroneous genesis. Thus, mutagenesis is not an essential aspect of SOS base pairing during DNA synthesis opposite the distorted repair. We will explain the effect of these novel mutants by lesion is responsible for the mutagenesis that accompanies showing that they are characterized by reactivation of UV SOS repair, and that idea has evoked the term error-prone lesions in a time too short for deamination to occur. repair. We propose, instead, that most, though certainly not One of us has proposed a theory of the mechanism of all, ofthe mutations arise through accurate base pairing-i.e., Weigle reactivation (12) that is sometimes called the A-rule. an error-free bypass mechanism. This paradox will be re- It was proposed in order to explain the specificity of SOS solved by the fact that mutations can arise by deamination of mutagenesis of UV-irradiated DNA, which was first demon- cytosine in cyclobutane dimers; ifthe uracils formed are read strated in phage S13 (13); since the TT dimer is expected to be the major dimer formed, it is notable that the major The publication costs of this article were defrayed in part by page charge mutational change is nevertheless C -* T. That striking result payment. This article must therefore be hereby marked "advertisement" could be explained if the SOS system blindly inserts adenine in accordance with 18 U.S.C. §1734 solely to indicate this fact. nucleotides opposite pyrimidine dimers regardless ofthe base 1159 Downloaded by guest on September 28, 2021 1160 Genetics: Tessman et al. Proc. Natl. Acad. Sci. USA 89 (1992) composition of the dimers, a process that would ensure RESULTS proper pairing for most of the dimerized sites. A similar A-rule has been proposed to explain the successful bypass of The experimental approach was simple. The latent period apurinic/apyrimidinic sites in DNA (14, 15). We will see that between infection and phage burst indicated the time delay associated with the of a lesion. We will see that the A-rule not only explains the C -- T specificity of SOS bypass mutagenesis, but that it also provides a tidy explanation for unirradiated phage S13 has a latent period of 15-20 min. Of the critical delay in the dimer bypass that is a key to the C this, <2 min is needed to synthesize the complement of the T mutations. infecting DNA molecule (20). In the case of UV-damaged DNA we make the reasonable assumption that once the lesion is bypassed and the complement completely synthe- MATERIALS AND METHODS sized, the phage will require no more than an additional 15-20 Strains and Medium. The bacterial strains used in this min to produce a burst. Therefore, ifL is the measured latent study were isogenic derivatives of the E. coli K-12 strain period ofreactivated phage, then L - 20 is the minimum time AB1157. All strains contained the sulAIl dinDl::Mu d(lac) needed for the bypass of the lesion. If this delay time is >30 alleles. The two high-copy-number plasmids pSE117 min, then deamination ofcytosines containing a saturated 5,6 (umuD+C+) and pGW2123 (umuD) were provided by G. C. bond should occur. Walker (4, 16). The original groES30 strain was obtained We first measured the latent period of unirradiated S13 in from C. P. Georgopolous (University of Utah). The chromo- some of the strains of bacteria that were used for Weigle somal recAl202 and recAl237 strains were constructed by reactivation and found (Table 1, lines 1-3) that the latent crossing the ArecA1202 and ArecA1237 phages with a recA' period was between 15 and 20 min, in agreement with A(gal-attA-bio) strain. All ArecA lysogens contained the previous data (22). The latent period of irradiated phage chromosomal ArecA306 allele (5); the ArecAl237 strains reactivated in strain IT1993, which contains constitutively also activated RecA, was between 115 and 130 min (line 4). We IT3663 and IT3665 contained the IexA(Def)71::TnS al- infer that the delay before the initial bypass occurred was lele. Tryptone broth contained 13 g of Bacto-tryptone and 7 between 95 and 110 min, which should allow adequate time g of NaCl per liter. for dimerized cytosines to be deaminated and thereby be a Determination of Latent Periods for Phages S13 and A. To major source of Weigle mutagenesis. The degree of muta- measure replication delay, bacterial cells were grown in genesis is quantitatively represented in Table 1 by the specific tryptone broth (with 0.2% maltose in the A experiments) to mutation frequency, Ms, which is defined as the number of =4 x 108 per ml.