Active-site determination of a pyrimidine dimer glycosylase

One mechanism for the repair of UV-induced DNA damage is the base excision repair pathway. The initial step in this pathway and the specificity for the type of damage that is to be repaired reside in DNA glycosylase/abasic (AP) lyases. Cleavage of the glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer is hypothesized to occur through the destabilization of the glycosyl bond by protonation of the base or sugar with a concomitant nucleophilic attack on C1' of the deoxyribose moiety. Based on mechanistic biochemical information from several glycosylase/AP lyases and the structural information on the bacteriophage T4 pyrimidine dimer glycosylase (T4-pdg), the catalytic mechanism has been investigated for the Chlorella virus pyrimidine dimer glycosylase (cv-pdg). As predicted from modeling studies and reaction mechanisms, the primary amine that initiates the nucleophilic displacement reaction could be trapped as a covalent imine intermediate and its identity determined by sequential Edman degradation. The primary amine was identified as the alpha-amino group on the N-terminal Thr2. Site-directed mutagenesis was subsequently used to confirm the conclusions that the alpha-amino group of cv-pdg is the active-site nucleophile.     

Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA

The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane pyrimidine dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 pyrimidine dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the alpha-amino group of the N-terminal threonine residue and the C1' of the deoxyribose sugar of the 5' pyrimidine at the dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA-protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing pyrimidine dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (approximately 66 degrees ) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.     

Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase.

The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.     

Uncoupling of nucleotide flipping and DNA bending by the t4 pyrimidine dimer DNA glycosylase

Bacteriophage T4 pyrimidine dimer glycosylase (T4-Pdg) is a base excision repair protein that incises DNA at cyclobutane pyrimidine dimers that are formed as a consequence of exposure to ultraviolet light. Cocrystallization of T4-Pdg with substrate DNA has shown that the adenosine opposite the 5'-thymine of a thymine-thymine (TT) dimer is flipped into an extrahelical conformation and that the DNA backbone is kinked 60 degrees in the enzyme-substrate (ES) complex. To examine the kinetic details of the precatalytic events in the T4-Pdg reaction mechanism, investigations were designed to separately assess nucleotide flipping and DNA bending. The fluorescent adenine base analogue, 2-aminopurine (2-AP), placed opposite an abasic site analogue, tetrahydrofuran, exhibited a 2.8-fold increase in emission intensity when flipped in the ES complex. Using the 2-AP fluorescence signal for nucleotide flipping, kon and koff pre-steady-state kinetic measurements were determined. DNA bending was assessed by fluorescence resonance energy transfer using fluorescent donor-acceptor pairs located at the 5'-ends of oligonucleotides in duplex DNA. The fluorescence intensity of the donor fluorophore was quenched by 15% in the ES complex as a result of an increased efficiency of energy transfer between the labeled ends of the DNA in the bent conformation. Kinetic analyses of the bending signal revealed an off rate that was 2.5-fold faster than the off rate for nucleotide flipping. These results demonstrate that the nucleotide flipping step can be uncoupled from the bending of DNA in the formation of an ES complex.     

Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase

The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location.     

den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA.

Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a DNA glycosylase activity that produced alkali-labile sites in irradiated DNA and an apyrimidinic/apurinic endonuclease activity that converted these sites to nicks. Both activities could be detected in the presence of 10 mM EDTA. In experiments designed to determine which of the activities is coded by the denV gene, we found that the glycosylase was more heat labile in extracts of Escherichia coli infected with either of two thermosensitive denV mutants than in extracts of cells infected with wild-type T4. In contrast, apyrimidinic/apurinic endonuclease activity was no more heat labile in extracts of the former than in extracts of the latter. Our results indicate that the denV gene codes for a DNA glycosylase specific for pyrimidine dimers.     

den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities.

Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.     

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.     

Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage

Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204–213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5–10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.     

Modulation of the turnover of formamidopyrimidine DNA glycosylase

In recent years, significant progress has been made in determining the catalytic mechanisms by which base excision repair (BER) DNA glycosylases and glycosylase-abasic site (AP) lyases cleave the glycosyl bond. While these investigations have identified active site residues and active site architectures, few investigations have analyzed postincision turnover events. Previously, we identified a critical residue (His16) in the T4-pyrimidine dimer glycosylase (T4-Pdg) that, when mutated, interferes with enzyme turnover [Meador et al. (2004) J. Biol. Chem. 279, 3348-3353]. To test whether comparable residues and mechanisms might be operative for other BER glycosylase:AP-lyases, molecular modeling studies were conducted comparing the active site regions of T4-Pdg and the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg). These analyses revealed that His71 in Fpg might perform a similar function to His16 in T4-Pdg. Site-directed mutagenesis of the Fpg gene and analyses of the reaction mechanism of the mutant enzyme revealed that the H71A enzyme retained activity on a DNA substrate containing an 8-oxo-7,8-dihydroguanine (8-oxoG) opposite cytosine and DNA containing an AP site. The H71A Fpg mutant was severely compromised in enzyme turnover on the 8-oxoG-C substrate but had turnover rates comparable to that of wild-type Fpg on AP-containing DNA. The similar mutant phenotypes for these two enzymes, despite a complete lack of structural or sequence homology between them, suggest a common mechanism for the rate-limiting step catalyzed by BER glycosylase:AP-lyases.     

Role of active site residues in the glycosylase step of T4 endonuclease V. Computer simulation studies on ionization states.

T4 Endonuclease V (EndoV) is a base excision repair enzyme that removes thymine dimers (TD) from damaged DNA. To elucidate the role of the active site residues in catalysis, their pK(a)'s were evaluated using the semimicroscopic version of the protein dipoles-Langevin dipoles method (PDLD/S). Contributions of different effects to the pK(a) such as charge-charge interactions, conformational rearrangement, protein relaxation, and DNA binding were analyzed in detail. Charging of the active site residues was found to be less favorable in the complex than in the free enzyme. The pK(a) of the N-terminus decreased from 8.01 in the free enzyme to 6.52 in the complex, while the pK(a) of Glu-23 increased from 1. 52 to 7.82, which indicates that the key residues are neutral in the reactant state of the glycosylase step. These pK(a)'s are in agreement with the optimal pH range of the reaction and support the N-terminus acting as a nucleophile. The Glu-23 in its protonated form is hydrogen bonded to O4' of the sugar of 5' TD and can play a role in increasing the positive charge of C1' and, hence, accelerating the nucleophilic substitution. Furthermore, the neutral Glu-23 is a likely candidate to protonate O4' to induce ring opening required to complete the glycosylase step of EndoV. The positively charged Arg-22 and Arg-26 provide an electrostatically favorable environment for the leaving base. To distinguish between S(N)1 and S(N)2 mechanisms of the glycosylase step the energetics of protonating O2 of 5' TD was calculated. The enzyme was found to stabilize the neutral thymine by approximately 3.6 kcal/mol, whereas it destabilizes the protonated thymine by approximately 6.6 kcal/mol with respect to an aqueous environment. Consequently, the formation of a protonated thymine intermediate is unlikely, indicating an S(N)2 reaction mechanism for the glycosylase step.     

Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo

The 3,N(4)-ethenocytosine (epsilon C) residue might have biological role in vivo since it is recognized and efficiently excised in vitro by the E. coli mismatch-specific uracil-DNA glycosylase (MUG) and the human thymine-DNA glycosylase (hTDG). In the present work we have generated mug defective mutant of E. coli by insertion of a kanamycin cassette to assess the role of MUG in vivo. We show that human TDG complements the enzymatic activity of MUG when expressed in a mug mutant. The epsilon C-DNA glycosylase defective strain did not exhibit spontaneous mutator phenotype and did not show unusual sensitivity to any of the following DNA damaging treatments: methylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet light, H(2)O(2), paraquat. However, plasmid DNA damaged by 2-chloroacetaldehyde treatment in vitro was inactivated at a greater rate in a mug mutant than in wild-type host, suggesting that MUG is required for the in vivo processing of the ethenobases. In addition, 2-chloroacetaldehyde treatment induces preferentially G.C --> C.G and A.T --> T.A transversions in mug mutant. Comparison of the mutation frequencies induced by the site-specifically incorporated epsilon C residue in E. coli wild-type versus mug indicates that MUG repairs more than 80% of epsilon C residues in vivo. Furthermore, the results show that nucleotide excision repair and recombination are not involved in the processing of epsilon C in E. coli. Based on the mutagenesis data we suggest that epsilon C may be less toxic and less mutagenic than expected. The increased spontaneous mutation rate for G.C --> A.T transition in the ung mug double mutant as compared to the single ung mutant suggest that MUG may be a back-up repair enzyme to the classic uracil-DNA glycosylase.     

Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study, we tested the role of excised base on MPG enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1,N⁶-ethenoadenine (?A) from ?A-containing oligonucleotide (?A-DNA) at a similar or slightly increased efficiency than it does hypoxanthine (Hx) from Hx-containing oligonucleotide (Hx-DNA) under similar conditions. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that both the substrate DNAs have a similar equilibrium binding constant (K_D) towards MPG, but under single-turnover (STO) condition there is apparently no effect on catalytic chemistry; however, the turnover of the enzyme under multiple-turnover (MTO) condition is higher for ?A-DNA than it is for Hx-DNA. Real-time binding experiments by SPR spectroscopy further showed that the dissociation of MPG from its product, AP-site containing DNA, is faster than the overall turnover of either Hx- or ?A-DNA reaction. We thereby conclude that the excised base plays a critical role in product inhibition and, hence, is essential for MPG glycosylase activity. Thus, the results provide the first evidence that the excised base rather than AP-site could be rate-limiting for DNA-glycosylase reactions.     

Light-induced activation of class II cyclobutane pyrimidine dimer photolyases

Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD photolyase and (6–4) photolyase. By ENDOR spectroscopy, the local environment of the flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FAD^ox, have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD photolyase, light-induced generation of FADH from FAD^ox, and subsequently FADH^? from FADH, proceeds in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I photolyases, but completely lacking from the equivalent positions of class II photolyase sequences. Nevertheless, class II photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution.     

Effects of DNA looping on pyrimidine dimer formation.

We have assessed the effects of DNA curvature on pyrimidine dimer (PD) formation by examining the pattern of PD formation in DNA held in a loop by lambda repressor. The loop region was composed of diverse DNA sequences such that potential PD sites occurred throughout the loop. PD formation in the loop occurred with peaks at approximately 10 base intervals, just 3' of where the bending of the DNA was inferred to be toward the major groove. This relationship between the peaks and the DNA curvature is essentially identical to that observed in the nucleosome. This indicates that DNA curvature is the major source of the periodicity of PD formation in the nucleosome, and supports an earlier model of the conformation of nucleosomal DNA based on PD formation. DNA loops containing diverse sequences should be of general value for assessing the effects of DNA curvature on DNA modification by other agents used to probe DNA-protein interactions and DNA conformation.     

Evidence that the UV endonuclease activity induced by bacteriophage T4 contains both pyrimidine dimer-DNA glycosylase and apyrimidinic/apurinic endonuclease activities in the enzyme molecule.

We performed experiments to determine whether the phage T4-induced UV endonuclease activity is a single protein containing both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities. The UV endonuclease activity is induced by the denV gene and codes for the glycosylase activity. We obtained several kinds of evidence that the protein containing the glycosylase activity also contains the apyrimidinic endonuclease activity. After chromatography on DEAE-cellulose, the two activities copurified during phosphocellulose chromatography and Sephadex G-100 chromatography, with a constant ratio of activities across the activity peaks. On Sephadex G-100 columns the molecular weights of the two activities agreed within 2,500 or less. When an extract of cells infected with the T4 V1 mutant was purified in exactly the same way as an extract of cells infected with T4 V1+, neither glycosylase nor apyrimidinic endonuclease activity was detected in the normal elution position of the T4 UV endonuclease activity. The glycosylase and apyrimidinic endonuclease activities were induced with similar kinetics, which were characteristic of immediate early rather than delayed early enzymes. This correlated well with the presumed major role of these activities in repairing thymine dimers in parental DNA before DNA replication begins. Finally, glycosylase and apyrimidinic endonuclease activities were lost in parallel during incubation of the enzyme at 46 degree C. Our results indicated that both of these enzyme activities are contained in the same enzyme molecule and, probably, in the same polypeptide.     

Properties of a UV-sensitive Neurospora strain defective in pyrimidine dimer excision

The UV-sensitive Neurospora strain uvs-2 is known to resemble the excision-defective uvr mutants of E. coli K12 in being both excision-defective and highly UV mutable. As shown in this report, the uvs-2 strain also resembles the uvr mutants in its ability to remain photoreactivable when held in the dark for 2 h between UV-irradiation and photoreactivating light exposure, and in its maintenance of the same spontaneous deletion rate as wild type strains.Unlike the E. coli uvr mutants, however, this strain is sensitive to ionizing radiation and shows an increase in survival when held for 2 h in distilled water before plating (liquid-holding recovery [LHR]). The strain is three times more sensitive to X-rays than the wild type strain. It is also sensitive to nitrosoguanidine (MNNG). Sensitivity to UV, X-rays and MNNG appears to be under the control of a single gene.These properties suggest that the repair defect in the Neurospora uvs-2 mutant is different from those of the uvr mutants of E. coli K12.     

Efficiency of pyrimidine dimer formation in Escherichia coli across UV wavelengths

AIMS: Inactivation of Escherichia coli as a function of ultraviolet (UV) wavelength was investigated by using the endonuclease-sensitive site (ESS) assay to quantify pyrimidine dimer formation. METHODS AND RESULTS: Ultraviolet dose-response curves were determined based on both log reduction in colony-forming units (CFU) and endonuclease-sensitive sites per kb DNA (ESS/kb) for monochromatic 254-nm low-pressure (LP) UV, polychromatic medium-pressure (MP) UV, 228 and 289-nm UV irradiation. UV irradiation from LP and MP UV sources were approx. equal in both CFU reduction and pyrimidine dimer formation at all UV doses studied; 228-nm irradiation was less effective than LP or MP, and 289-nm irradiation was the least effective in both CFU reduction and pyrimidine dimer formation. These results are in qualitative agreement with the absorption spectrum of pyrimidine bases in DNA. Results indicated an approx. linear relationship between ESS/kb and log CFU reduction. CONCLUSIONS: Formation of pyrimidine dimers in genomic DNA is primarily responsible for UV inactivation of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributed to fundamental understanding of UV disinfection and aids in UV reactor design.     

Role of pyrimidine dimer excision in loss of potential streptomycin resistance mutations of ultraviolet-irradiated Escherichia coli on phosphate-buffered agar.

The frequency of ultraviolet (UV)-induced mutations to streptomycin resistance dropped rapidly when starved Escherichia coli strains WP-2 B/r and B/r T- were incubated on phosphate-buffered agar (PBA), but was reduced only slightly in a WP-2 hcr- mutant. During postirradiation, incubation viability remained approximately constant. Cells given an optimal recovery treatment with photo-reactivating light showed no further recovery if subsequently incubated on PBA. At least 70% of the mutations induced to streptomycin resistance by UV could be repaired. The loss of potential streptomycin-resistant mutants was markedly reduced in strain B/r T- when 5 mug of acriflavin or 700 mug of caffeine per ml was added to PBA. The excision of UV-induced thymine-containing dimers from E. coli tb/r T- was investigated. Dimer excision progressed more slowly when the cells were incubated on PBA containing acriflavin or caffeine. There was no congruity between the kinetics of dimer excision and the kinetics of mutant loss. Our results indicate that removal of potential streptomycin-resistant mutants is considerably faster than the excision of pyrimidine dimers.