UV Lesions Research Articles

Yeast RAD26, a homolog of the human CSB gene, functions independently of nucleotide excision repair and base excision repair in promoting transcription through damaged bases.

RAD26 in the yeast Saccharomyces cerevisiae is the counterpart of the human Cockayne syndrome group B (CSB) gene. Both RAD26 and CSB act in the preferential repair of UV lesions on the transcribed strand, and in this process, they function together with the components of nucleotide excision repair (NER). Here, we examine the role of RAD26 in the repair of DNA lesions induced upon treatment with the alkylating agent methyl methanesulfonate (MMS). MMS-induced DNA lesions include base damages such as 3-methyl adenine and 7-methyl guanine, and these lesions are removed in yeast by the alternate competing pathways of base excision repair (BER), which is initiated by the action of MAG1-encoded N-methyl purine DNA glycosylase, and NER. Interestingly, a synergistic increase in MMS sensitivity was observed in the rad26 Delta strain upon inactivation of NER or BER, indicating that RAD26 promotes the survival of MMS-treated cells by a mechanism that acts independently of either of these repair pathways. The galactose-inducible transcription of the GAL2, GAL7, and GAL10 genes is reduced in MMS-treated rad26 Delta cells and also in mag1 Delta rad14 Delta cells, whereas a very severe reduction in transcription occurs in MMS-treated mag1 Delta rad14 Delta rad26 Delta cells. From these observations, we infer that RAD26 plays a role in promoting transcription by RNA polymerase II through damaged bases. The implications of these observations are discussed in this paper.

Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae.

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.

Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6 H,8 H-pyrimido[4,5- c][1,2]oxazin-7-one 2′-deoxyriboside (dP), which is highly mutagenic to bacteria. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6–4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1 +. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3–5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.

An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates.

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.

Requirement for yeast RAD26, a homolog of the human CSB gene, in elongation by RNA polymerase II.

Mutations in the human CSB gene cause Cockayne syndrome (CS). In addition to increased photosensitivity, CS patients suffer from severe developmental abnormalities, including growth retardation and mental retardation. Whereas a deficiency in the preferential repair of UV lesions from the transcribed strand accounts for the increased photosensitivity of CS patients, the reason for developmental defects in these individuals has remained unclear. Here we provide in vivo evidence for a role of RAD26, the counterpart of the CSB gene in Saccharomyces cerevisiae, in transcription elongation by RNA polymerase II, and in addition we show that under conditions requiring rapid synthesis of new mRNAs, growth is considerably reduced in cells lacking RAD26. These findings implicate a role for CSB in transcription elongation, and they strongly suggest that impaired transcription elongation is the underlying cause of the developmental problems in CS patients.

Accuracy of lesion bypass by yeast and human DNA polymerase eta.

DNA polymerase eta (Pol eta) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Pol eta in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Pol eta replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Pol eta, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of approximately 10(-2) to 10(-3) opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Pol eta in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Pol eta to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Pol eta extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of approximately 10(-2) to 10(-3) relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Pol eta would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3'-->5' exonuclease. Replication through a TT dimer by Pol eta then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

ATP-dependent chromatin remodeling facilitates nucleotide excision repair of UV-induced DNA lesions in synthetic dinucleosomes.

To investigate the relationship between chromatin dynamics and nucleotide excision repair (NER), we have examined the effect of chromatin structure on the formation of two major classes of UV-induced DNA lesions in reconstituted dinucleosomes. Furthermore, we have developed a model chromatin-NER system consisting of purified human NER factors and dinucleosome substrates that contain pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) either at the center of the nucleosome or in the linker DNA. We have found that the two classes of UV-induced DNA lesions are formed efficiently at every location on dinucleosomes in a manner similar to that of naked DNA, even in the presence of histone H1. On the other hand, excision of 6-4PPs is strongly inhibited by dinucleosome assembly, even within the linker DNA region. These results provide direct evidence that the human NER machinery requires a space greater than the size of the linker DNA to excise UV lesions efficiently. Interestingly, NER dual incision in dinucleosomes is facilitated by recombinant ACF, an ATP-dependent chromatin remodeling factor. Our results indicate that there is a functional connection between chromatin remodeling and the initiation step of NER.

Two-Wavelength Fluorescence Assay for DNA Repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.

Cover Picture

The cover picture shows the sun together with a damaged (back) and repaired (front) DNA strand. The emitted sun energy is the basis for all life on earth. The UV part of the electromagnetic spectrum, however, causes the formation of a variety of mutagenic DNA lesions, which endanger the integrity of the genetic material. These lesions are repaired by a class of photolyases which utilize long-wavelength sunlight and a flavin coenzyme to initiate a critical electron transfer from the enzyme to the UV lesion. Carell and co-workers describe on p. 3918 ff the synthesis of lesion-containing DNA strands in which the flavin coenzyme was integrated. These DNA strands show a sunlight-driven self-repair process based on a surplus electron transfer through the base stack. (The sun picture is courtesy of the SOHO consortium; SOHO–Solar and Heliospheric Observatory—is a project of international cooperation between ESA and NASA.)

Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro.

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

Alteration of ultraviolet-induced mutagenesis in yeast through molecular modulation of the REV3 and REV7 gene expression.

DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition. Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein. Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.

Resolution of holliday junctions by RuvABC prevents dimer formation in rep mutants and UV-irradiated cells.

In this work, we present evidence that indicates that RuvABC proteins resolve Holliday junctions in a way that prevents dimer formation in vivo. First, although arrested replication forks are rescued by recombinational repair in cells deficient for the Rep helicase, rep mutants do not require the XerCD proteins or the dif site for viability. This shows that the recombination events at arrested replication forks are generally not accompanied by the formation of chromosome dimers. Secondly, resolution of dimers into monomers is essential in the rep ruv strain because of an increased frequency of RecFOR recombination events in the chromosome of this mutant. This suggests that, in the absence of the Ruv proteins, chromosomal recombination leads to frequent dimerization. Thirdly, dif or xerC mutations increase the UV sensitivity of ruv-deficient cells 100-fold, whereas they do not confer UV sensitivity to ruv+ cells. This shows that recombinational repair of UV lesions is not accompanied by dimer formation provided that the RuvABC proteins are active. The requirement for dimer resolution in ruv strains is suppressed by the expression of the RusA Holliday junction resolvase; therefore, RusA also prevents dimer formation. We conclude that the inviability arising from a high frequency of dimer formation in rep or UV-irradiated cells is only observed in the absence of known enzymes that resolve Holliday junctions.

Specific amino acid changes enhance the anti-recombination activity of the UmuD'C complex.

In addition to being an essential component of trans-lesion synthesis, the UmuD'C complex is an antagonist of RecA-mediated homologous recombination. When constitutively expressed at an elevated concentration, the UmuD'C complex sensitizes recA+ bacteria to DNA damage, whereas it has no effect on bacteria expressing a RecA [UmuR] protein that overcomes recombination inhibition. Using as a genetic screen enhanced cell killing on mitomycin plates, we isolated novel umuD' and umuC mutations that restored mitomycin sensitivity to recA D112G [UmuR] bacteria overproducing the UmuD'C complex. The mutations were named [Rin++] because a characterization in a recA+ as well in a recA D112G background showed that they enhanced UmuD'C-promoted recombination inhibition in two assays, conjugational recombination and recombinational repair of palindrome-containing DNA. The [Rin++] mutations affect five amino acids, G25D, S28T, P29L, E35K, and T95R, in UmuD' and seven, F10L, Y270C, K277E, F287L, F287S, K342Q and F351I, in UmuC. These amino acids might play a key role in the UmuD'C anti-recombination activity. None of the [Rin++] mutations enhanced UmuD'C-promoted mutagenic bypass of UV lesions, in contrast, several lead to a defect in this process. In this study, we discuss a few molecular mechanisms that could account for the recombination and mutagenesis phenotypes of a mutant UmuD'C [Rin++] complex.

Inducible stable DNA replication (iSDR) and the uvr-dependent tolerance of pyrimidine dimers in UV-irradiated Escherichia coli are two uncoupled processes

Inducible stable DNA replication (iSDR) is dependent on recombination and is supposed to play a role in DNA repair of Escherichia coli . Our previous data suggested that iSDR may be involved in the tolerance of UV lesions, which remain unexcised in excision-proficient E. coli exposed to some UV pretreatments. Now, the tolerance of unexcised lesions has been followed in E. coli recB21 and in E. coli priA1 sup mutants, incapable of iSDR. The obtained data do not confirm the previous hypothesis about the involvement of iSDR in the putative uvr-dependent lesion tolerance. They rather suggest that iSDR and the uvr-dependent lesion tolerance are two uncoupled processes.

UV lesions located on the leading strand inhibit DNA replication but do not inhibit SV40 T-antigen helicase activity

DNA replication in eucaryotic cells involves a variety of proteins which synthesize the leading and lagging strands in an asymmetric coordinated manner. To analyse the effect of this asymmetry on the translesion synthesis of UV-induced lesions, we have incubated SV40 origin-containing plasmids with a unique site-specific cis,syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct on either the leading or lagging strand template with DNA replication-competent extracts made from human HeLa cells. Two dimensional agarose gel electrophoresis analyses revealed a strong blockage of fork progression only when the UV lesion is located on the leading strand template. Because DNA helicases are responsible for unwinding duplex DNA ahead of the fork and are then the first component to encounter any potential lesion, we tested the effect of these single photoproducts on the unwinding activity of the SV40 T antigen, the major helicase in our in vitro replication assay. We showed that the activity of the SV40 T-antigen helicase is not inhibited by UV-induced DNA lesions in double-stranded DNA substrate.

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6–4) pyrimidones (6–4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6–4PD, can lead to cell killing and mutations.

Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair.

Nucleotide excision repair (NER) and DNA repair by photolyase in the presence of light (photoreactivation) are the major pathways to remove UV-induced DNA lesions from the genome, thereby preventing mutagenesis and cell death. Photoreactivation was found in many prokaryotic and eukaryotic organisms, but not in mammals, while NER seems to be universally distributed. Since packaging of eukaryotic DNA in nucleosomes and higher order chromatin structures affects DNA structure and accessibility, damage formation and repair are coupled intimately to structural and dynamic properties of chromatin. Here, I review recent progress in the study of repair of chromatin and transcribed genes. Photoreactivation and NER are discussed as examples of how an individual enzyme and a complex repair pathway, respectively, access DNA lesions in chromatin and how these two repair processes fulfil complementary roles in removal of UV lesions. These repair pathways provide insight into the structural and dynamic properties of chromatin and suggest how other DNA repair processes could work in chromatin.

SOS mutagenesis results from up-regulation of translesion synthesis

Irradiation of DNA with ultraviolet light generates a variety of photolesions. Among them, are cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts blocking lesions that interfere with DNA replication if left unrepaired. In addition to efficient pre-replicative excision repair mechanisms, cells have evolved damage tolerance pathways enabling them to replicate lesion-containing DNA molecules either by directly replicating through the damaged base (translesion synthesis, TLS) or by employing the locally undamaged complementary strand thus avoiding the lesion (damage avoidance pathways, DA). Using double-stranded vectors with a single T(6-4)T UV lesion and a strand segregation analysis (SSA), we have measured the relative utilization of the two tolerance pathways (TLS and DA) in Escherichia coli. During the SOS response the error-prone TLS pathway is strongly stimulated (≈20-fold) at the expense of the error-free DA pathways. Thus, up-regulation of TLS may turn out to be a general property of the SOS response; a similar conclusion was previously reached with the frameshift-inducing N-2-acetylaminofluorene adduct. Therefore, as far as its contribution to damaged DNA replication is concerned, the SOS response appears to be an induced mutator state rather than a survival strategy. Depending on the base inserted opposite the lesion, TLS can be error-free or mutagenic. In a wild-type strain, both forms of TLS are increased to a similar extent during the SOS response. In contrast, in a Δ umuDC strain induction of TLS is totally abolished, demonstrating that the UmuDC proteins usually thought to be specifically involved in mutagenesis facilitate the recovery of both error-free and mutagenic replication intermediates in vivo.

Urocanic Acid Photochemistry and Photobiology

Urocanic acid (2-propenoic acid, 3-[ lH-imidazol-4(5)-yl], UA)? is one of the smallest molecules to have stimulated global interest among biologists, environmentalists, photochemists, photobiologists, medicinal chemists and immunologists. Its history can be traced back more than a century to when it was first found in the urine of dogs (1). Interest in the molecule remained dormant until the middle of the century but rekindled in the late 1940s when it was detected in animal skin and sweat. This led to the proposal that UA acts as a natural sunscreen, possibly as a specific photoprotecting agent for DNA because of the overlap of the UA and DNA absorption spectra (2). Since 1983 there has been an explosive growth in UA research, primarily as a consequence of the proposal by De Fabo and Noonan (3) that the cis photoisomer (cUA) of trans-UA (tUA) could be responsible for the phenomenon of photoimmunosuppression. In a recent literature survey we found that about 25 research papers have appeared every year over the last decade involving UA. Several comprehensive reviews of the molecule's photobiology, photochemistry and photophysics have appeared (4-

Nucleotide excision repair: From E. coli to man

Nucleotide excision repair is both a `wide spectrum' DNA repair pathway and the sole system for repairing bulky damages such as UV lesions or benzo[a]pyrene adducts. The mechanisms of nucleotide excision repair are known in considerable detail in Escherichia coli. Similarly, in the past 5 years important advances have been made towards understanding the biochemical mechanisms of excision repair in humans. The overall strategy of the repair is the same in the two species: damage recognition through a multistep mechanism involving a molecular matchmaker and an ATP-dependent unwinding of the damaged duplex; dual incisions at both sides of the lesion by two different nucleases, the 3' incision being followed by the 5'; removal of the damaged oligomer; resynthesis of the repair patch, whose length matches the gap size. Despite these similarities, the two systems are biochemically different and do not even share structural homology. E. coli excinuclease employs three proteins in contrast to 16/17 polypeptides in man; the excised fragment is longer in man; the procaryotic excinuclease is not able by itself to remove the excised oligomerwhereas the human enzyme does. Thus, the excinuclease mode of action is well conserved throughout evolution, but not the biochemical tools: this represents a case of evolutionary convergence.