Milan Skorvaga

Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism. NER systems recognize the damaged DNA strand, cleave it on both sides of the lesion, remove and newly synthesize the fragment. UvrB is a central component of the bacterial NER system participating in damage recognition, strand excision and repair synthesis. We have solved the crystal structure of UvrB in the apo and the ATP‐bound forms. UvrB contains two domains related in structure to helicases, and two additional domains unique to repair proteins. The structure contains all elements of an intact helicase, and is evidence that UvrB utilizes ATP hydrolysis to move along the DNA to probe for damage. The location of conserved residues and structural comparisons allow us to predict the path of the DNA and suggest that the tight pre‐incision complex of UvrB and the damaged DNA is formed by insertion of a flexible β‐hairpin between the two DNA strands.

Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage

Telomerase is often re‐activated in human cancers and is widely used to immortalize cells in culture. In addition to the maintenance of telomeres, telomerase has been implicated in cell proliferation, genomic instability and apoptosis. Here we show that human telomerase reverse transcriptase (hTERT) is targeted to the mitochondria by an N‐terminal leader sequence, and that mitochondrial extracts contain telomerase activity. In seven different human cell lines, mitochondrial telomerase increases hydrogen‐peroxide‐mediated mitochondrial DNA damage. hTERT expression did not alter the rate of hydrogen peroxide breakdown or endogenous cellular levels. Because the damaging effects of hydrogen peroxide are mediated by divalent metal ions (Fenton chemistry), we examined the levels of bioavailable metals. In all cases, higher levels of chelatable metals were found in hTERT‐expressing cells. These results suggest that mitochondrial telomerase sensitizes cells to oxidative stress, which can lead to apoptotic cell death, and imply a novel function of telomerase in mitochondrial DNA transactions.

Structural insights into the first incision reaction during nucleotide excision repair.

Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3′ incision reaction. Here we report the crystal structure of the N‐terminal catalytic domain of UvrC at 1.5 Å resolution, which catalyzes the 3′ incision reaction and shares homology with the catalytic domain of the GIY‐YIG family of intron‐encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium‐water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY‐YIG endonuclease domains. Structural and biochemical data strongly suggest that the N‐terminal endonuclease domain of UvrC utilizes a novel one‐metal mechanism to cleave the phosphodiester bond.

Interactions between UvrA and UvrB: the role of UvrB's domain 2 in nucleotide excision repair.

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism present in all kingdoms of life. UvrB is a central component of the bacterial NER system, participating in damage recognition, strand excision and repair synthesis. None of the three presently available crystal structures of UvrB has defined the structure of domain 2, which is critical for the interaction with UvrA. We have solved the crystal structure of the UvrB Y96A variant, which reveals a new fold for domain 2 and identifies highly conserved residues located on its surface. These residues are restricted to the face of UvrB important for DNA binding and may be critical for the interaction of UvrB with UvrA. We have mutated these residues to study their role in the incision reaction, formation of the pre‐incision complex, destabilization of short duplex regions in DNA, binding to UvrA and ATP hydrolysis. Based on the structural and biochemical data, we conclude that domain 2 is required for a productive UvrA–UvrB interaction, which is a pre‐requisite for all subsequent steps in nucleotide excision repair.

Repair activity of base and nucleotide excision repair enzymes for guanine lesions induced by nitrosative stress.

Nitric oxide (NO) induces deamination of guanine, yielding xanthine and oxanine (Oxa). Furthermore, Oxa reacts with polyamines and DNA binding proteins to form cross-link adducts. Thus, it is of interest how these lesions are processed by DNA repair enzymes in view of the genotoxic mechanism of NO. In the present study, we have examined the repair capacity for Oxa and Oxa–spermine cross-link adducts (Oxa–Sp) of enzymes involved in base excision repair (BER) and nucleotide excision repair (NER) to delineate the repair mechanism of nitrosative damage to guanine. Oligonucleotide substrates containing Oxa and Oxa–Sp were incubated with purified BER and NER enzymes or cell-free extracts (CFEs), and the damage-excising or DNA-incising activity was compared with that for control (physiological) substrates. The Oxa-excising activities of Escherichia coli and human DNA glycosylases and HeLa CFEs were 0.2–9% relative to control substrates, implying poor processing of Oxa by BER. In contrast, DNA containing Oxa–Sp was incised efficiently by UvrABC nuclease and SOS-induced E.coli CFEs, suggesting a role of NER in ameliorating genotoxic effects associated with nitrosative stress. Analyses of the activity of CFEs from NER-proficient and NER-deficient human cells on Oxa–Sp DNA confirmed further the involvement of NER in the repair of nitrosative DNA damage.

UvrB Domain 4, an Autoinhibitory Gate for Regulation of DNA Binding and ATPase Activity

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBΔ4) leads to multiple changes in protein function. Protein dimerization decreases with an ∼8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBΔ4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBΔ4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.

Transfection of the Escherichia coli nth Gene into Radiosensitive Chinese Hamster Cells: Effects on Sensitivity to Radiation, Hydrogen Peroxide, and Bleomycin Sulfate

The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA. A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7. Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene. No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1). However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity [D0s for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0s of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively] but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0s for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0s for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively). Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines. These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic. Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival. Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival. The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.

Incidence of Common Preleukemic Gene Fusions in Umbilical Cord Blood in Slovak Population

The first event in origination of many childhood leukemias is likely the presence of preleukemic clone (transformed hematopoietic stem/progenitor cells with preleukemic gene fusions (PGF)) in newborn. Thus, the screening of umbilical cord blood (UCB) for PGF may be of high importance for developing strategies for childhood leukemia prevention and treatment. However, the data on incidence of PGF in UCB are contradictive. We have compared multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (RT qPCR) in neonates from Slovak National Birth Cohort. According to multiplex PCR, all 135 screened samples were negative for the most frequent PGF of B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To explore the prevalence of prognostically important TEL-AML1, MLL-AF4 and BCR-ABL (p190), 200 UCB were screened using RT qPCR. The initial screening showed an unexpectedly high incidence of studied PGF. The validation of selected samples in two laboratories confirmed approximately ¼ of UCB positive, resulting in ∼4% incidence of TEL-AML1, ∼6.25% incidence of BCR-ABL1 p190, and ∼0.75% frequency of MLL-AF4. In most cases, the PGF presented at very low level, about 1–5 copies per 105 cells. We hypothesize that low PGF numbers reflect their relatively late origin and are likely to be eliminated in further development while higher number of PGF reflects earlier origination and may represent higher risk for leukemia.

Reduced sulfhydryls maintain specific incision of BPDE-DNA adducts by recombinant thermoresistant Bacillus caldotenax UvrABC endonuclease.

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. Escherichia coli UvrABC endonuclease can incise DNA containing UV photoproducts and bulky chemical adducts. The limited stability of the E. coli UvrABC subunits leads to difficulty in estimating incision efficiency and quantitative adduct detection. To develop a more stable enzyme with greater utility for the detection of DNA adducts, thermoresistant UvrABC endonuclease was cloned from the eubacterium Bacillus caldotenax (Bca) and individual recombinant protein subunits were overexpressed in and purified from E. coli. Here, we show that Bca UvrC that had lost activity or specificity could be restored by dialysis against buffer containing 500 mM KCl and 20mM dithiothreitol. Our data indicate that UvrC solubility depended on high salt concentrations and UvrC nuclease activity and the specificity of incisions depended on the presence of reduced sulfhydryls. Optimal conditions for BCA UvrABC-specific cleavage of plasmid DNAs treated with [3H](+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (1-5 lesions/plasmid) were developed. Preincubation of substrates with UvrA and UvrB enhanced incision efficiency on damaged substrates and decreased non-specific nuclease activity on undamaged substrates. Under optimal conditions for damaged plasmid incision, approximately 70% of adducts were incised in 1 nM plasmid DNA (2 BPDE adducts/5.4 kbp plasmid) with UvrA at 2.5 nM, UvrB at 62.5 nM, and UvrC at 25 nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.

Effect of expression of the Escherichia coli nth gene in Saccharomyces cerevisiae on the toxicity of ionizing radiation and hydrogen peroxide.

Purpose: To examine the contribution of endonuclease III (Nth)‐repairable lesions to the cytotoxicity of ionizing radiation (IR) and hydrogen peroxide (H2O2) in the yeast Saccharomyces cerevisiae. Materials and methods: A selectable expression vector containing the E. coli nth gene was transformed into two different wild‐type strains (7799‐4B and YNN‐27) as well as one rad52 mutant strain (C5–6). Nth expression was verified by Western analysis. Colony‐forming assay was used to determine the sensitivity to IR and H2O2 in both stationary and exponentially growing cells. Results: The pADHnth‐transformed wild‐type (7799‐4B) strain was considerably more resistant than vector‐only transformants to the toxic effects of IR, in both stationary and exponential growth phases, although this was not the case in another wild‐type strain (YNN‐27). In contrast, there were no significant effects of nth expression on the sensitivity of the wild‐type cells to H2O2. Moreover, nth expression caused no effects on the H2O2 sensitivity in the rad52 mutant cells, but it led to a slight increase in sensitivity in these cells following IR, particularly at the highest dose levels used. Conclusions: Whilst other damage‐processing systems may play a role, DNA lesions that are substrates for Nth can also make a contribution to the toxic effects of IR in certain wild‐type yeast. Hence, DNA double‐strand breaks should not be considered the sole lethal lesions following IR exposure.