Characterization of molecular defects in xeroderma pigmentosum group C.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease of humans characterized by an accelerated chronic degeneration of sun-exposed areas of the body, including an elevated risk of developing cancers of the skin. We recently reported the isolation of a gene XPCC that complements the repair deficiency of cultured XP-C cells. Here we report the results of a characterization of XPCC at the nucleotide level in five XP-C cell lines. Each cell line exhibited a unique mutation that correlated well with the cellular DNA repair deficiency and the clinical severity of the disease. These results extend our previous observations and indicate that defects in XPCC cause Xeroderma pigmentosum group C.

Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair.

The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as delta G and delta E. In vitro, the binding of ERCC1 to delta E was reduced by approximately 70%, and binding to delta G was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the delta E mutant exhibited an intermediate level of complementation of XPA cells and the delta G mutant exhibited little or no complementation. In addition, the delta G mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The delta E and delta G mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damage site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min.

Interstrand cross-links induce DNA synthesis in damaged and undamaged plasmids in mammalian cell extracts.

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.

Repair of UV-induced lesions in Xenopus laevis oocytes.

We characterized a DNA repair system in frog oocytes by comicroinjection of UV-irradiated pBR322 DNA and radiolabeled nucleotides. Repair synthesis was monitored by incorporation of label into recovered pBR322 DNA and by a novel method in which the removal of UV photoproducts was determined from the shift of DNA topoisomers that occurs during gel electrophoresis upon repair of these lesions. We investigated the effects of several drugs in the oocyte system and found that although novobiocin, an inhibitor of topoisomerase II, was an effective inhibitor of repair, VM-26, another inhibitor of topoisomerase II, was not. In addition, the topoisomerase I inhibitor camptothecin had no effect on repair in this system. Finally, circular DNA (either supercoiled or nicked circular) was repaired at least 50 times more rapidly than linear DNA.

An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential.

Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair.

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

Requirement for PCNA and RPA in interstrand crosslink-induced DNA synthesis.

Proliferating nuclear cell antigen (PCNA) and replication protein A (RPA) have proven to be essential elements in many aspects of DNA metabolism including replication, repair and recombination. We have developed an in vitro assay in which the presence of an interstrand crosslink stimulates the incorporation of radiolabeled nucleotides into both damaged and undamaged plasmid DNAs. Using this assay we have investigated the roles of PCNA and RPA in crosslink-induced DNA synthesis. p21, a potent inhibitor of PCNA, was found to strongly inhibit crosslink-induced incorporation. Addition of exogenous PCNA partially restored the resynthesis activity. Likewise, neutralization of RPA by monoclonal antibodies also inhibited incorporation, but the effect was somewhat more pronounced on the undamaged plasmid than the damaged plasmid. Addition of excess RPA also partially reversed antibody inhibition. These results indicate that both PCNA and RPA are required for efficient in vitro DNA resynthesis induced by interstrand crosslinks.

Simultaneous amplification of four DNA repair genes and beta-actin in human lymphocytes by multiplex reverse transcriptase-PCR.

We describe here the development, optimization, and use of a non-radioactive, quantitative, multiplex reverse transcriptase-PCR technique to measure, in a single reaction, the relative levels of the transcripts of four DNA repair genes (XPCC, hMSH2, XRCC1, and ERCC1) and the beta-actin gene in lymphoblastoid cell lines and frozen peripheral blood lymphocytes. Expression of defective DNA repair genes was not detected in DNA repair-deficient human cell lines, whereas the intact genes were detected in repair-proficient cell lines and in lymphocytes from a normal donor. The assay was reproducible, and repeated determinations of the same samples generated highly consistent results for each target gene. This approach should facilitate molecular epidemiological studies that incorporate screening for germline alterations that may affect gene expression and for changes in the levels of gene expression.

hRAD17, a structural homolog of the Schizosaccharomyces pombe RAD17 cell cycle checkpoint gene, stimulates p53 accumulation.

The RAD17 gene product of S. Pombe is an essential component of the checkpoint control pathway which responds to both DNA damage and disruption of replication. We have identified a human cDNA that encodes a polypeptide which is structurally conserved with the S. Pombe Rad17 protein. The human gene, designated hRAD17, predicts an encoded protein of 590 amino acids and a molecular weight of 69 kD. Amino acid sequence alignment revealed that hRadl7 has 28.3% and 52.5% similarity with the S. Pombe Rad17 protein, and 21.8% identity and 45.8% similarity to the budding yeast cell cycle checkpoint protein, Rad 24. When introduced into the S. Pombe rad17 mutant, hRAD17 was able to partially revert its hydroxyurea and ionizing radiation hypersensitivity, but not its UV hypersensitivity. Permanent overexpression of the hRAD17 gene in human fibrosarcoma cells resulted in p53 activation and a significant reduction of S- and G2/M-phase cells accompanied by an accumulation of the G1-phase population, suggesting that hRAD17 may have a role in cell cycle checkpoint control. Immunostaining of HT-1080 cells transiently transfected with a hRAD17 construct confirmed the nuclear accumulation of p53, which mimics the induction caused by DNA damage. Using FISH analysis, we have mapped the hRAD17 locus to human chromosome 5q11.2.

A sedimentation velocity method for the separation of complementary strands of DNA.

A method is described for the rapid separation of the complementary strands of homogeneous DNA's. The method takes advantage of a difference in sedimentation coefficients between the complementary strands when these have been dissociated and complexed with poly(U-G). The separation procedure is readily carried out for preparative purposes by zone sedimentation in linear 5--20% (w/v) sucrose gradients containing 2 M NaCl. In analytical band sedimentation experiments, concentrated solutions of CsCl have been used to effect the separation of the complexed complementary strands. Sedimentation coefficients corresponding to corrected values at infinite dilution for the cesium salt of the nucleic acid complexes at 25 degrees C (s 25, w) have been obtained for the complexed complementary strands of coliphage lambdab2b5c DNA and for the uncomplexed single strands of this DNA. The relative values of the parameters affecting s 25, w have been determined. Under these conditions, for which preferential hydration of the sedimenting species is not involved, it was found that the more highly complexed strand has sustained a 24% increase in mass and a 28% decrease in frictional coefficient relative to uncomplexed DNA, while the strand binding the lesser amount of RNA has increased in mass by 11% and changed in frictional coefficient by an insignificant amount relative to the uncomplexed species from that of the uncomplexed DNA do not contribute significantly to the differences in s 25, w between the complexed complementary strands.

Evidence for a salt-induced conformational transition in UV-irradiated superhelical PM2 DNA.

Upon treatment with UV irradiation, native (supercoiled) PM2 DNA undergoes an increase in electrophoretic mobility relative to the nicked circular form in the presence of 1 M NaCl or 5 mM CaCl2 or MgCl2. This effect is dependent upon supercoiling in that the relative electrophoretic mobility decreases with decreasing superhelical density of the molecule. These findings indicate that supercoil-dependent aspects of the secondary and tertiary structure of nonirradiated PM2 DNA can be altered by a combination of UV irradiation and any of the ionic environments above. We show that the alteration is not the result of a conversion of Z-DNA segments to a right-handed helix or to a renaturation of denatured regions in PM2 DNA. Circular dichroism studies do not support a simple model in which A-form DNA induced by superhelical stress is converted to B-form DNA by UV-induced photodamage and salt. We, therefore, present three alternative explanations for these observations two of which invoke conformational transitions in secondary structure and a third which requires a change in tertiary structure due to an increase in flexibility.

Type I DNA topoisomerases from mammalian cell nuclei interlock strains and promote renaturation of denatured closed circular PM2 DNA.

Type I DNA topoisomerases from mouse ascites cell nuclei and from rat liver cell nuclei act on denatured viral closed circular PM2 DNA to produce molecules with a highly contracted structure as well as fully duplex non-supercoiled covalently closed circular molecules. Highly contracted DNA molecules contain a novel type of topological linkage in which a strand in one region of the double-stranded molecule passes between the strands in another region of the circular molecule one or more times. Since it is also found that the action of the topoisomerase promotes renaturation of complementary strands in denatured closed circular DNA, it is suggested that formation of contracted DNA structures proceeds through renatured, duplex intermediates with highly negative superhelix densities that contain small single-stranded regions.

A single apurinic site can elicit BAL 31 nuclease-catalyzed cleavage in duplex DNA.

The extracellular nuclease from Alteromonas espejiana BAL 31 is a highly sensitive endonucleolytic probe for lesions that distort the helical structure of duplex DNA. The nuclease can be isolated as two distinct molecular species, the 'fast' (F) and 'slow' (S) species, which have different kinetic properties. When nonsupercoiled, covalently closed circular phage PM2 DNA containing apurinic sites introduced by heating at acid pH was incubated with individual fractions from a chromatographic column which separated the two nuclease species, cleavage of the DNA was observed which was greatly in excess of control levels using nonmodified DNA. The initial rates of such cleavage clearly paralleled the peaks of both absorbance and nuclease activity against single-stranded and linear duplex substrates. When samples of apurinic DNA were incubated with pooled fractions from the same column representing pure F and S nucleases, respectively, the rate and extent of the cleavage observed was dependent upon the average number of apurinic sites per molecule. Cleavage was readily detectable in samples containing an average of 1.1 apurinic sites per molecule with both species of the enzyme. These results indicate that either species of the BAL 31 nuclease can recognize and cleave in response to a single apurinic site in duplex DNA. The F nuclease appears to be approx. 2.5-times as efficient in cleaving DNA containing apurinic lesions as the S enzyme in extended incubations.

Analysis and optimization of recombinant DNA joining reactions.

The statistical segment length of duplex DNA was determined in phage T4 ligase (poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMP forming), EC 6.5.1.1) buffer (50 mM-Tris . HCl (pH 7.8), 20 mM-dithiothreitol, 10 mM-MgCl2, 1 mM-ATP) at 12 degrees C to be 1030(+/- 116) A. This result was obtained by electron microscopic examination of the molecular distributions generated by T4 ligase-mediated joining of EcoRI-cleaved pBR322 DNA. This value of the statistical segment length was utilized in an extension of the Jacobson-Stockmayer theory on the probability of intramolecular cyclization in order to optimize DNA joining reactions that are of great utility in recombinant DNA experiments. Five cloning systems were analyzed: circular plasmid vectors that had been linearized with one or two restriction endonucleases, circular plasmids that had been tailed with deoxyhomopolymers before joining, lambda-type cloning vectors and cosmids. The results are tabulated for convenient use in molecular cloning experiments.

Expression in normal human tissues of five nucleotide excision repair genes measured simultaneously by multiplex reverse transcription-polymerase chain reaction.

DNA repair is central to the integrity of the human genome. Reduced DNA repair capacity has been linked to genetic susceptibility to cancer. An adequate expression level of DNA repair genes is essential for normal DNA repair activities. Although there is tissue specificity in the expression, searching for a surrogate tissue is needed for molecular epidemiological studies. In this study, the relative expression levels of five selected human nucleotide excision repair (NER) genes (ERCC1, XPB/ERCC3, XPG/ERCC5, CSB/ERCC6, and XPC) in 20 different types of human normal tissue were simultaneously measured by a new multiplex reverse transcription (RT)-PCR assay using the expression level of the beta-actin gene as an internal control. Transcripts of each of the five NER genes were detectable, but the levels varied in these normal tissues. Both mitogen (phytohemagglutinin)-stimulated and unstimulated human peripheral lymphocytes showed similar expression patterns for the five NER genes. In general, the expression levels of stimulated lymphocytes were also similar to most of the rapidly proliferating tissues, such as the skin, breast, intestine, liver, testis, ovary, placenta, or prostate, but was relatively higher than that of the slowly proliferating or nonproliferating tissues such as adipose, brain, hippocampus, muscle, spleen, or lung. The data suggested that although the five NER genes were expressed at different levels in the normal tissues examined, PHA-stimulated peripheral lymphocytes may be used as a surrogate tissue for estimating expression levels of these genes in proliferating tissues. This new multiplex RT-PCR assay may help detect aberrant expression of these NER genes in both normal and tumor tissues.

Cellular responses to ionizing radiation damage.

PURPOSE: The purpose of this report is to provide current perspectives on studies of DNA damage and cell cycle response after ionizing radiation, and their applications in radiation oncology. METHODS AND MATERIALS: Presentations at the Seventh Annual Radiation Oncology Workshop, held at the International Festival Institute at Round Top, TX, were summarized. RESULTS: Eighteen speakers presented their current work covering a wide range of studies on cellular responses to ionizing radiation. These presentations and discussions form the framework of our report. CONCLUSION: In response to ionizing radiation, cells immediately activate a series of biochemical pathways that promote cell survival while maintaining genetic integrity. The main cellular defense system against ionizing radiation exposure is composed of two distinct types of biochemical pathways, that is, the DNA damage cell cycle checkpoint pathways and the DNA repair pathways. The DNA damage checkpoint pathways are activated directly by DNA damage, while the repair pathways are constitutively active and are likely modulated by checkpoint signals. Discussions here emphasize that the ATM protein is a central component of the ionizing radiation-responsive pyramid and is essential for activating divergent molecular responses that involve transcriptional regulation, cell cycle arrest, and modulation of DNA repair. The relationship between homologous recombinational repair and nonhomologous end joining of double-strand breaks is also discussed.

Removal of 2',3'-dideoxynucleotide residues from injected DNA in Xenopus laevis oocytes.

Dideoxynucleotides have proved to be potent differential inhibitors of DNA polymerases in vitro and in vivo. Used extensively in studies of DNA repair and replication, they have more recently been used as antiviral agents particularly in treating patients for acquired immunodeficiency syndrome (AIDS). Once incorporated, these sugar-modified analogues prevent the further extension of the polynucleotide chain because of the absence of a 3'-hydroxyl group. We demonstrated that, upon injection into Xenopus laevis oocytes, 2',3'-dideoxynucleotides are efficiently removed from plasmid DNA preterminated with these analogues allowing subsequent closure by ligation. The removal process is not sensitive to aphidicolin but is quantitatively inhibited by novobiocin.

Artemis is a negative regulator of p53 in response to oxidative stress.

Artemis is a multifunctional phospho-protein with roles in V(D)J recombination, repair of double-strand breaks by nonhomologous end-joining and regulation of cell-cycle checkpoints after DNA damage. Here, we describe a new function of Artemis as a negative regulator of p53 in response to oxidative stress in both primary cells and cancer cell lines. We show that depletion of Artemis under typical culture conditions (21% oxygen) leads to a spontaneous phosphorylation and stabilization of p53, and resulting cellular G1 arrest and apoptosis. These effects are suppressed by co-depletion of DNA-PKcs, but not ATM, indicating that Artemis is an inhibitor of DNA-PKcs-mediated stabilization of p53. Culturing of cellsat 3% oxygen or treatment with an antioxidant abrogated p53 stabilization, indicating that oxidative stress is the responsible cellular stimulus. Treatment with ionizing radiation or hydrogen peroxide did not cause activation of this signaling pathway, whereas inhibitors of mitochondrial electron transport were effective in reducing its activation. In addition, we show that p53-inducible genes involved in reducing reactive oxygen species are upregulated by Artemis depletion. These findings indicate that Artemis and DNA-PKcs participate in a new, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.

Snm1B/Apollo mediates replication fork collapse and S Phase checkpoint activation in response to DNA interstrand cross-links.

The removal of DNA interstrand cross-links (ICLs) has proven to be notoriously complicated due to the involvement of multiple pathways of DNA repair, which include the Fanconi anemia/BRCA pathway, homologous recombination and components of the nucleotide excision and mismatch repair pathways. Members of the SNM1 gene family have also been shown to have a role in mediating cellular resistance to ICLs, although their precise function has remained elusive. Here, we show that knockdown of Snm1B/Apollo in human cells results in hypersensitivity to mitomycin C (MMC), but not to IR. We also show that Snm1B-deficient cells exhibit a defective S phase checkpoint in response to MMC, but not to IR, and this finding may account for the specific sensitivity to the cross-linking drug. Interestingly, although previous studies have largely implicated ATR as the major kinase activated in response to ICLs, we show that it is activation of the ATM-mediated checkpoint that is defective in Snm1B-deficient cells. The requirement for Snm1B in ATM checkpoint activation specifically after ICL damage is correlated with its role in promoting double-strand break formation, and thus replication fork collapse. Consistent with this result Snm1B was found to interact directly with Mus81-Eme1, an endonuclease previously implicated in fork collapse. In addition, we also show that Snm1B interacts with the Mre11-Rad50-Nbs1 (MRN) complex and with FancD2 further substantiating its role as a checkpoint/DNA repair protein.

A sensitive endonuclease probe for lesions in deoxyribonucleic acid helix structure produced by carcinogenic or mutagenic agents.

The highly single strand-specific extracellular nuclease of Pseudomonas BAL 31 is shown to cleave non-supercoiled closed circular duplex PM2 bacteriophage DNA containing regions of altered helix structure produced in vitro by irradiation with ultraviolet light or by treatment with the carcinogen, N-acetoxy-N-2-acetylaminofluorene. Untreated samples of this DNA are affected very little by the nuclease. The unwinding of the DNA helix associated with the above treatment renders the closed circular DNA positively supercoiled compared to untreated samples. The extent of unwinding can be accurately measured and correlated with the average number of lesions per molecule of DNA by monitoring the alterations of the electrophoretic patterns, relative to those observed for untreated DNA, of such DNA in agarose gels. Interstrand cross-links and mismatched base pairs produced by treatment of non-supercoilded circular duplex DNA with the mutagen, nitrous acid, do not detectably unwind the DNA helix. The nitrous acid-treated DNA provides substrates for cleavage by the Pseudomonas nuclease which are likely to be the interstrand cross-links rather than the mismatched base pairs. Use of the Pseudomonas nuclease in conjunction with agarose gel electrophoresis can provide a powerful method for the detection of damage in duplex DNA such as that introduced by carcinogenic and mutagenic agents.