Although sunlight is essential for life on earth, the ultraviolet (UV) wavelengths in its spectrum constitute a major threat to life. Various cellular responses have evolved to deal with the damage inflicted in DNA by UV, and the study of these responses in model systems has spawned the burgeoning field of DNA repair. Although we now know of many types of deleterious alterations in DNA, the approaches for studying them and the early mechanistic insights have come in large part from pioneering research on the processing of UV-induced bipyrimidine photoproducts in bacteria. It is also notable that UV was one of the first DNA damaging agents for which exposure was directly linked to cancer; the sun-sensitive syndrome, xeroderma pigmentosum, was the first example of a cancer-prone hereditary disease involving a defect in DNA repair. We provide a short history of advances in the broad field of genomic maintenance as they have emerged from research in photochemistry and photobiology.

A quarter of a century has elapsed since the discovery of transcription-coupled repair (TCR), and yet our fascination with this process has not diminished. Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. TCR, defined as a subpathway of NER, is dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, we will report on newly identified proteins, protein modifications, and protein complexes that participate in TCR in Escherichia coli and in human cells. We will discuss general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.

When Joshua Lederberg (known to friends and colleagues as Josh) died on 2 February 2008, the world lost one of the most extraordinary scientists of the twentieth century. It is difficult to write an adequate memorial for him, or to convey on paper the outpouring of admiration and affection expressed in written and oral presentations by experts in widely diverse fields of inquiry. Even listing his various interests and achievements is a formidable task, and adequately evaluating the importance of his contributions to science and society is almost impossible. As the founding father of bacterial genetics, Lederberg was the first to demonstrate the conjugal transfer of genetic markers in bacteria. Together with his associates, he went on to make many more discoveries that laid the foundations of molecular genetics. For this work he received the 1958 Nobel Prize in Physiology or Medicine, sharing the award with G. W. Beadle and E. L. Tatum. While retaining an interest in bacterial genetics, he went on to explore and make seminal contributions to numerous other disciplines including exobiology (a term he coined), the application of computers and artificial intelligence to chemistry and medicine, and the epistemology of science. He advised US presidents and international organizations on a wide variety of issues, and devoted a prodigious amount of time and effort to the task of informing policy makers and the larger public on important scientific matters. Joshua Lederberg was a man not only of towering intellect but also of impeccable integrity and dedication to human welfare.

Transcription-coupled nucleotide excision repair (TC-NER) removes certain kinds of lesions from the transcribed strand of expressed genes. The signal for TC-NER is thought to be RNA polymerase stalled at a lesion in the DNA template. In Escherichia coli, the stalled polymerase is dissociated from the lesion by the transcription repair coupling factor (Mfd protein), which also recruits excision repair proteins to the site resulting in efficient removal of the lesion. TC-NER has been documented in cells from a variety of organisms ranging from bacteria to humans. In each case, the RNA polymerase involved has been a multimeric protein complex. To ascertain whether a gene transcribed by the monomeric RNA polymerase of bacteriophage T7 could be repaired by TC-NER, we constructed strains of E. coli in which the chromosomal lacZ gene is controlled by a T7 promoter. In the absence of T7 RNA polymerase, little or no β-galactosidase is produced, indicating that the E. coli RNA polymerase does not transcribe lacZ efficiently, if at all, in these strains. By introducing a plasmid (pAR1219) carrying the T7 gene 1 under control of the E. coli lac UV5 promoter into these strains, we obtained derivatives in which the level of T7 RNA polymerase could be regulated. In cultures containing upregulated levels of the polymerase, β-galactosidase was actively produced indicating that the T7 RNA polymerase transcribes the lacZ gene efficiently. Under these conditions, we observed that UV-induced cyclobutane pyrimidine dimers were removed more rapidly from the transcribed strand of lacZ than from the nontranscribed strand, supporting the conclusion that TC-NER occurred in this gene. This response was absent in an mfd-1 mutant, indicating that the underlying mechanism may be similar to that for the bacterial RNA polymerase.

The proposed mechanism for transcription coupled nucleotide excision repair (TCR) invokes RNA polymerase (RNAP) blocked at a DNA lesion as a signal to initiate repair. In Escherichia coli, TCR requires the interaction of RNAP with a transcription-repair coupling factor encoded by the mfd gene. The interaction between RNAP and Mfd depends upon amino acids 117, 118, and 119 of the beta subunit of RNAP; changing any one of these to alanine diminishes the interaction [1]. Using direct assays for TCR, and the lac operon of E. coli containing UV induced cyclobutane pyrimidine dimers (CPDs) as substrate, we have found that a change from arginine to cysteine at amino acid 529 of the beta subunit of the RNAP inactivates TCR, but does not prevent the interaction of RNAP with Mfd. Our results suggest that this interaction may be necessary but not sufficient to facilitate TCR.

The order of discovery can have a profound effect upon the way in which we think about the function of a gene. In E. coli, recA is nearly essential for cell survival in the presence of DNA damage. However, recA was originally identified, as a gene required to obtain recombinant DNA molecules in conjugating bacteria. As a result, it has been frequently assumed that recA promotes the survival of bacteria containing DNA damage by recombination in which DNA strand exchanges occur. We now know that several of the processes that interact with or are controlled by recA, such as excision repair and translesion synthesis, operate to ensure that DNA replication occurs processively without strand exchanges. Yet the view persists in the literature that recA functions primarily to promote recombination during DNA repair. With the benefit of hindsight and more than three decades of additional research, we reexamine some of the classical experiments that established the concept of DNA repair by recombination, and we consider the possibilities that recombination is not an efficient mechanism for rescuing damaged cells, and that recA may be important for maintaining processive replication in a manner that does not generally promote recombination.

In the thirty-third Cold Spring Harbor Symposium, mycolleagues and I reported on our demonstration of "Repair replication of DNA in vivo" (Hanawalt et al. 1969).We began with the cautious statement, "It has been postulated that damaged DNA in bacteria and in some othercellular systems is subject to repair by a mechanism thatinvolves the replacement of the defective single-strandregions with a short stretch of oligonucleotide utilizingthe complementary base pairing information in the intactstrand." That symposium was held just a few years following the discovery of excision repair in Escherichiacoli (Boyce and Howard-Flanders 1964; Pettijohn andHanawalt 1964; Setlow and Carrier 1964). At that meeting, we also learned of an endonuclease activity in extracts of T4-bacteriophage-infected E. coli that incisesUV-irradiated DNA (Takagi et al. 1969). The responsibleenzyme, later designated T4 endoV, was to figure prominently in our research in subsequent years as a highly specific and sensitive probe for UV-induced cyclobutanepyrimidine dimers (CPDs) (Ganesan 1973; van Zeelandet al. 1981; Bohr et al. 1985). The fledgeling DNA repairfield received an important stimulus that same year withthe discovery that repair replication is defective in cellsfrom victims of the rare autosomal recessive hereditarydisease, xeroderma pigmentosum (XP), characterized bysevere sensitivity to sunlight and a several thousandfoldincrease in cutaneous cancers (Cleaver 1968). For manyyears thereafter, it has been considered that nucleotideexcision repair (NER) is of importance primarily for dealing with UV-induced DNA photoproducts—a view reinforced by the fact that the incidence of internal cancers inXP patients is not remarkably enhanced...

It has been suggested that reactivation of damaged reporter genes introduced into cultured mammalian cells reflects transcription-coupled nucleotide excision repair. To evaluate this possibility directly, we introduced a UV-irradiated shuttle vector, pCMVβ, into unirradiated human cells and compared expression of the reporter gene (lacZ) with repair of cyclobutane pyrimidine dimers (CPDs). Expression of the irradiated reporter gene was more UV resistant in XPC cells, which are deficient in global genome repair, than in CSB cells, which are deficient in transcription-coupled repair. These results are consistent with the idea that repair of the reporter gene is primarily dependent upon transcription-coupled repair. However, when the plasmid DNA was analyzed for removal of CPDs, no clear evidence was obtained for transcription-coupled repair either in XPC cells or in cells with normal repair capacity.

A shuttle vector (pZH-1) carrying the E. coli lacZ gene under control of the SV40 early promoter was irradiated with UV and introduced into repair-proficient or repair-deficient human cell lines. The expression of irradiated lacZ compared to unirradiated lacZ was greater in repair-proficient cells (HT-1080) than in repair-deficient cells (XP12RO-SV40) belonging to xeroderma pigmentosum complementation group A. To ascertain whether the expression of lacZ in the repair-proficient cells was correlated with the removal of cyclobutane pyrimidine dimers (CPDs), we purified DNA from the recipient cells and used the CPD-specific enzyme T4 endonuclease V to measure the frequency of CPDs remaining in the plasmid as a whole and in two restriction fragments derived from it. We found that removal of CPDs occurred in both fragments in the repair-proficient cells but not in the repair-deficient cells. Our results provide the first direct evidence for the removal of CPDs from UV irradiated plasmids introduced into human cells and support the notion that expression of the UV-damaged lacZ gene in repair-proficient human cells reflects the removal of transcription blocking lesions from the gene.

We have found that several excision deficient derivatives of Escherichia coli K12 survive better after UV irradiation if incubated at 42°C than if incubated at 30°C. The highest survival was observed when incubation at 42°C followed UV irradiation and was maintained for at least 16 h. Our results indicate that this temperature dependent resistance (TDR) requires a functional recA gene, but not uvr A, uvrB, recF, or recB genes, or the recA441 (tif-1) mutation which allows thermoinduction of the recA-lexA regulon. Our data are consistent with the idea that the increase in survival observed at 42°C reflects enhanced daughterstrand gap repair by DNA strand exchange. Although the conditions used to elicit TDR can induce heat shock proteins and thermotolerance in E. coli, the relationship between the two responses remains to be elucidated.

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.

We have shown that when pSV2-gpt is introduced into human cells by calcium phosphate coprecipitation, the yield of Gpt+ transformants is increased by irradiating the plasmid with 254 nm uv. To elucidate the mechanism underlying this response, we constructed pSV2-gpt molecules in which the uv damage was confined to a particular region: a 3.0-kb region containing the pBR322 sequences and simian virus 40 (SV40) sequences not required for expression of the gpt gene, or a 2.3-kb fragment containing the Escherichia coli gpt gene together with the SV40 early promoter and sequences needed for splicing and polyadenylation. The transforming activity of the plasmid was greatly enhanced by uv damage confined to the 3.0-kb pBR322 region and increased linearly with uv dose up to 1 kJ/m2, but remained relatively constant at doses between 2 and 8 kJ/m2. Positioning the damaged region upstream, or both upstream and downstream, from the gpt transcription unit increased the uv enhancement slightly compared to positioning the damaged region only downstream. In contrast, transforming activity was significantly decreased by damage in the 2.3-kb gpt transcription unit. These results suggest that uv damage outside a selectable marker gene in a plasmid can increase the probability of stable integration of the plasmid into the genome of recipient cells without inhibiting expression of of the gene.

We previously reported that endonuclease V of bacteriophage T4 reacts processively with pyrimidine dimers in UV-irradiated DNA, tending to react with all of the dimers on one DNA molecule before reacting with any dimers on another DNA molecule [Lloyd, R. S., Hanawalt, P. C., & Dodson, M. L. (1980) Nucleic Acids Res. 8, 5113-5127]. In this paper we show that this processivity depends upon salt concentration: it can be detected in 10 mM NaCl but not, by our methods, in 100 mM NaCl. In addition, we show that endonuclease V binds to unirradiated DNA in 10 mM NaCl but not in 100 mM NaCl. We conclude that T4 endonuclease V binds to pyrimidine dimers in a two-step process in 10 mM NaCl. It first binds electrostatically to undamaged sections of DNA, and it remains bound during the second step in which it "searches" for pyrimidine dimers. Our conclusion is analogous to the expanded target theory developed for Lac repressor [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry 20, 6929-6948].

The lexA41 mutant of E. coli is a UV-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. lexA41 carried an additional mutation which changed amino acid 132 in the LexA protein from Ala to Thr. The resultant protein was unstable and was degraded both before and after an inducing treatment. This instability was greater at 42 degrees than at 30 degrees. The protein was more stable in Lon- mutants at both temperatures. lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistant with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the UV-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA- mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis.

Derivatives of Escherichia coli K-12 carrying a deletion of the recA gene survive exposure to UV (254 nm) better if they also contain the lexA41 mutation which codes for a labile LexA protein. This effect of the lexA41 mutation is not observed in comparable strains carrying a uvr A6 mutation. Using two independent methods to detect pyrimidine dimers we found that UV irradiated RecA deficient cells removed dimers from their DNA more rapidly if they contained the lexA41 mutation than if the contained the wild-type lexA gene. Our results are consistent with the idea that a relatively high level of UvrABC incision nuclease resulting from inefficient repression of the corresponding genes by the labile LexA41 protein facilitates excision of pyrimidine dimers from the DNA of UV irradiated cells.

Irradiating the plasmid pSV2-gpt with UV (254 nm) doses up to 200 J m-2 caused a dose-dependent increase in the yield of Gpt+ transformants when the plasmid was introduced into human cells by calcium phosphate coprecipitation. UV doses greater than 1 kJ m-2 were required to reduce the efficiency of transformation below that obtained with unirradiated DNA.

AbstractBacteria and eukaryotic cells employ a variety of enzymatic pathways to remove damage from DNA or to lessen its impact upon cellular functions. Most of these processes were discovered in Escherichia coli and have been most extensively analyzed in this organism because suitable mutants have been isolated and characterized. Analogous pathways have been inferred to exist in mammalian cells from the presence of enzyme activities similar to those known to be involved in repair in bacteria, from the analysis of events in cells treated with DNA damaging agents, and from the analysis of the few naturally occurring mutant cell types.Excision repair of pyrimidine dimers produced by UV in E coli is initiated by an incision event catalyzed by a complex composed of uvrA, uvrB, and uvrC gene products. Multiple exonuclease and polymerase activities are available for the subsequent excision and resynthesis steps. In addition to the constitutive pathway, which produces short patches of 20–30 nucleotides, an inducible excision repair process exists that produces much longer patches. This long patch pathway is controlled by the recA‐lexA regulatory circuit and also requires the recF gene. It is apparently not responsible for UV‐induced mutagenesis. However, the ability to perform inducible long patch repair correlates with enhanced bacterial survival and with a major component of the Weigle reactivation of bacteriophage with double‐strand DNA genomes.Mammalian cells possess an excision repair pathway similar to the constitutive pathway in E coli. Although not as well understood, the incision event is at least as complex, and repair resynthesis produces patches of about the same size as the constitutive short patches. In mammalian cells, no patches comparable in size to those produced by the inducible pathway of E coli are observed.Repair in mammalian cells may be more complicated than in bacteria because of the structure of chromatin, which can affect both the distribution of DNA damage and its accessibility to repair enzymes. A coordinated alteration and reassembly of chromatin at sites of repair may be required. We have observed that the sensitivity of digestion by staphylococcal nuclease (SN) of newly synthesized repair patches resulting from excision of furocoumarin adducts changes with time in the same way as that of patches resulting from excision of pyrimidine dimers. Since furocoumarin adducts are formed only in the SN‐sensitive linker DNA between nucleosome cores, this suggests that after repair resynthesis is completed, the nucleosome cores in the region of the repair event do not return exactly to their original positions.We have also studied excision repair of UV and chemical damage in the highly repeated 172 base pair α DNA sequence in African green monkey cells. In UV irradiated cells, the rate and extent of repair resynthesis in this sequence is similar to that in bulk DNA. However, in cells containing furocoumarin adducts, repair resynthesis in α DNA is only about 30% of that in bulk DNA. Since the frequency of adducts does not seem to be reduced in α DNA, it appears that certain adducts in this unique DNA may be less accessible to repair.Endonuclease V of bacteriophage T4 incises DNA at pyrimidine dimers by cleaving first the glycosylic bond between deoxyribose and the 5′ pyrimidine of the dimer and then the phosphodiester bond between the two pyrimidines. We have cloned the gene (denV) that codes for this enzyme and have demonstrated its expression in uvrA recA and uvrB recA cells of E coli. Because T4 endonuclease V can alleviate the excision repair deficiency of xeroderma pigmentosum when added to permeabilized cells or to isolated nuclei after UV irradiation, the cloned denV gene may ultimately be of value for analyzing DNA repair pathways in cultured human cells.

endonuclease activity thatconverted these sites tonicks. Bothactivities couldbedetected in thepresence of10mM EDTA.Inexperiments designed todetermine whichof theactivities iscodedbythedenVgene, wefoundthattheglycosylase wasmore heatlabile'in extracts ofEscherichia coli infected witheither oftwothermosen- sitive denVmutants thaninextracts ofcells infected withwild-type T4.In contrast, apyrimidinic/apurinic endonuclease activity wasnomoreheatlabile in extracts oftheformer thaninextracts ofthelatter. Ourresults indicate thatthe denVgenecodesforaDNA glycosylase specific forpyrimidine dimers. Until recently thedenVgeneofbacteriophage T4wasthought tocodeforanenzyme, desig- natedendonuclease V,thatspecifically incised UV-irradiated DNA bycleaving aphosphodies- terbondonthe5'sideofeachpyrimidine dimer (13, 17,21,23). However, anewmechanism of action hasrecently beenproposed forthisen- zymeandforanalogous enzymes fromMicro- coccus luteus basedupontheproperties offrag- mentsproduced bytreating UV-irradiated DNA ofknownnucleotide sequence withtheseen- zymes(8, 8a). Comparison oftheelectrophoretic mobilities ofthese fragments withthose offrag- mentsgenerated bychemical reactions that cleave DNA atspecific basesindicated that strand scission atthesiteofa dimerentails cleaving twobonds: theglycosylic bondofthe5' pyrimidine ofthedimer andthephosphodiester bondbetween thetwopyrimidines comprising thedimer. Wehaveobtained evidence that sup- ports theproposed schemeandindicates that thedenVgeneofT4codes fortheglycosylase activity.

Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme--DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. The assay is simple, rapid, and specific, which makes it useful for detecting T4 endonuclease V activity both in crude lysates and in purified preparations. We have used it to monitor enzyme activity during purification and to study binding of the enzyme to DNA under conditions that minimize the ability of the enzyme to nick DNA. From our data we conclude that (1) T4 endonuclease V binds to UV-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme--DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme--DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline--citrate.

Publisher Summary The chapter explains purification and properties of a pyrimidine dimer-specific endonuclease from E. coli infected with bacteriophage T4. The pyrimidine dimer-specific endonuclease of bacteriophage T4 is coded by the v (denV) gene. The enzyme has been called endonuclease V of phage T4 or the T4 uv endonuclease. For detecting pyrimidine dimers a preparation of enzyme free of activity against unirradiated DNA or DNA containing other types of damage is required. The chapter describes several methods for assaying T4 endonuclease V as well as the partial purification and general properties of the enzyme. The chapter discusses a reproducible procedure for obtaining stable preparations of the enzyme which can be used as a specific probe for pyrimidine dimers. For the purpose of purification, the enzyme can be assayed by one of three general methods— namely, coupled nuclease assay, filter binding assay, and DNA nicking assay. The chapter further discusses enzyme purification.