The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics.

Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1-RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression.

Alterations in c-Myc phenotypes resulting from dynamin-related protein 1 (Drp1)-mediated mitochondrial fission.

The c-Myc (Myc) oncoprotein regulates numerous phenotypes pertaining to cell mass, survival and metabolism. Glycolysis, oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis are positively controlled by Myc, with myc-/- rat fibroblasts displaying atrophic mitochondria, structural and functional defects in electron transport chain (ETC) components, compromised OXPHOS and ATP depletion. However, while Myc influences mitochondrial structure and function, it is not clear to what extent the reverse is true. To test this, we induced a state of mitochondrial hyper-fission in rat fibroblasts by de-regulating Drp1, a dynamin-like GTPase that participates in the terminal fission process. The mitochondria from these cells showed reduced mass and interconnectivity, a paucity of cristae, a marked reduction in OXPHOS and structural and functional defects in ETC Complexes I and V. High rates of abortive mitochondrial fusion were observed, likely reflecting ongoing, but ultimately futile, attempts to normalize mitochondrial mass. Cellular consequences included reduction of cell volume, ATP depletion and activation of AMP-dependent protein kinase. In response to Myc deregulation, apoptosis was significantly impaired both in the absence and presence of serum, although this could be reversed by increasing ATP levels by pharmacologic means. The current work demonstrates that enforced mitochondrial fission closely recapitulates a state of Myc deficiency and that mitochondrial integrity and function can affect Myc-regulated cellular behaviors. The low intracellular ATP levels that are frequently seen in some tumors as a result of inadequate vascular perfusion could favor tumor survival by countering the pro-apoptotic tendencies of Myc overexpression.

Impaired mitochondrial biogenesis contributes to depletion of functional mitochondria in chronic MPP+ toxicity: dual roles for ERK1/2.

The regulation of mitochondrial quality has emerged as a central issue in neurodegeneration, diabetes, and cancer. We utilized repeated low-dose applications of the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP(+)) over 2 weeks to study cellular responses to chronic mitochondrial stress. Chronic MPP(+) triggered depletion of functional mitochondria resulting in diminished capacities for aerobic respiration. Inhibiting autophagy/mitophagy only partially restored mitochondrial content. In contrast, inhibiting activation of extracellular signal-regulated protein kinases conferred complete cytoprotection with full restoration of mitochondrial functional and morphological parameters, enhancing spare respiratory capacity in MPP(+) co-treated cells above that of control cells. Reversal of mitochondrial injury occurred when U0126 was added 1 week after MPP(+), implicating enhanced repair mechanisms. Chronic MPP(+) caused a >90% decrease in complex I subunits, along with decreases in complex III and IV subunits. Decreases in respiratory complex subunits were reversed by co-treatment with U0126, ERK1/2 RNAi or transfection of dominant-negative MEK1, but only partially restored by degradation inhibitors. Chronic MPP(+) also suppressed the de novo synthesis of mitochondrial DNA-encoded proteins, accompanied by decreased expression of the mitochondrial transcription factor TFAM. U0126 completely reversed each of these deficits in mitochondrial translation and protein expression. These data indicate a key, limiting role for mitochondrial biogenesis in determining the outcome of injuries associated with elevated mitophagy.

Mitochondrially localized PKA reverses mitochondrial pathology and dysfunction in a cellular model of Parkinson's disease.

Mutations in PTEN-induced kinase 1 (PINK1) are associated with a familial syndrome related to Parkinson's disease (PD). We previously reported that stable neuroblastoma SH-SY5Y cell lines with reduced expression of endogenous PINK1 exhibit mitochondrial fragmentation, increased mitochondria-derived superoxide, induction of compensatory macroautophagy/mitophagy and a low level of ongoing cell death. In this study, we investigated the ability of protein kinase A (PKA) to confer protection in this model, focusing on its subcellular targeting. Either: (1) treatment with pharmacological PKA activators; (2) transient expression of a constitutively active form of mitochondria-targeted PKA; or (3) transient expression of wild-type A kinase anchoring protein 1 (AKAP1), a scaffold that targets endogenous PKA to mitochondria, reversed each of the phenotypes attributed to loss of PINK1 in SH-SY5Y cells, and rescued parameters of mitochondrial respiratory dysfunction. Mitochondrial and lysosomal changes in primary cortical neurons derived from PINK1 knockout mice or subjected to PINK1 RNAi were also reversed by the activation of PKA. PKA phosphorylates the rat dynamin-related protein 1 isoform 1 (Drp1) at serine 656 (homologous to human serine 637), inhibiting its pro-fission function. Mimicking phosphorylation of Drp1 recapitulated many of the protective effects of AKAP1/PKA. These data indicate that redirecting endogenous PKA to mitochondria can compensate for deficiencies in PINK1 function, highlighting the importance of compartmentalized signaling networks in mitochondrial quality control.

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures.

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.

The nucleotide excision repair protein UvrB, a helicase-like enzyme with a catch.

Nucleotide excision repair (NER) is a universal DNA repair mechanism found in all three kingdoms of life. Its ability to repair a broad range of DNA lesions sets NER apart from other repair mechanisms. NER systems recognize the damaged DNA strand and cleave it 3', then 5' to the lesion. After the oligonucleotide containing the lesion is removed, repair synthesis fills the resulting gap. UvrB is the central component of bacterial NER. It is directly involved in distinguishing damaged from undamaged DNA and guides the DNA from recognition to repair synthesis. Recently solved structures of UvrB from different organisms represent the first high-resolution view into bacterial NER. The structures provide detailed insight into the domain architecture of UvrB and, through comparison, suggest possible domain movements. The structure of UvrB consists of five domains. Domains 1a and 3 bind ATP at the inter-domain interface and share high structural similarity to helicases of superfamilies I and II. Not related to helicase structures, domains 2 and 4 are involved in interactions with either UvrA or UvrC, whereas domain 1b was implicated for DNA binding. The structures indicate that ATP binding and hydrolysis is associated with domain motions. UvrB's ATPase activity, however, is not coupled to the separation of long DNA duplexes as in helicases, but rather leads to the formation of the preincision complex with the damaged DNA substrate. The location of conserved residues and structural comparisons with helicase-DNA structures suggest how UvrB might bind to DNA. A model of the UvrB-DNA interaction in which a beta-hairpin of UvrB inserts between the DNA double strand has been proposed recently. This padlock model is developed further to suggest two distinct consequences of domain motion: in the UvrA(2)B-DNA complex, domain motions lead to translocation along the DNA, whereas in the tight UvrB-DNA pre-incision complex, they lead to distortion of the 3' incision site.

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA.

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.

Butadiene-induced intrastrand DNA cross-links: a possible role in deletion mutagenesis.

To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links, site specifically adducted oligodeoxynucleotides were synthesized in which the adducted bases were centrally located within the context of the human ras 12 codon. The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and transfected into Escherichia coli. Both stereoisomeric forms (R, R and S,S) of the DNA cross-links resulted in very severely decreased plaque-forming ability, along with an increased mutagenic frequency for both single base substitutions and deletions compared with unadducted DNAs, with the S,S stereoisomer being the most mutagenic. Consistent with decreased plaque formation, in vitro replication of DNA templates containing the cross-links by the three major E. coli polymerases revealed replication blockage by both stereoisomeric forms of the cross-links. The same DNAs that were used for replication studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E. coli UvrABC complex. UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently repaired by the nucleotide excision repair system.

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 beta-hairpin between the two DNA strands.

Bcl-2 facilitates recovery from DNA damage after oxidative stress.

Oxidative stress is a major factor affecting the brain during aging and neurodegenerative diseases such as Alzheimer's disease (AD). Understanding the mechanisms by which neurons can be protected from oxidative stress, therefore, is critical for the prevention and treatment of such degeneration. Previous studies have shown that bcl-2 expression is increased in neurons with DNA damage in AD and bcl-2 has an antioxidant effect. The goal of this study is to document the effects of oxidative insults on mitochondrial and nuclear DNA in PC12 cells and determine the extent to which bcl-2 prevents damage or facilitates repair. Using extralong PCR to amplify nuclear and mitochondrial DNA, the time course of DNA damage and repair was determined. Within minutes after exposure of cells to low concentrations of hydrogen peroxide and peroxynitrite, significant mitochondrial and nuclear DNA damage was evident. Mitochondrial DNA was damaged to a greater degree than nuclear DNA. Expression of bcl-2 in PC12 cells inhibited nitric oxide donor (sodium nitroprusside)- and peroxynitrite-induced cell death. Although oxidative insults caused both genomic and mitochondrial DNA damage in cells expressing bcl-2, recovery from DNA damage was accelerated in these cells. These results suggest that neuronal up-regulation of bcl-2 may facilitate DNA repair after oxidative stress.

Strand opening by the UvrA(2)B complex allows dynamic recognition of DNA damage.

Repair proteins alter the local DNA structure during nucleotide excision repair (NER). However, the precise role of DNA melting remains unknown. A series of DNA substrates containing a unique site-specific BPDE-guanine adduct in a region of non-complementary bases were examined for incision by the Escherichia coli UvrBC endonuclease in the presence or absence of UvrA. UvrBC formed a pre-incision intermediate with a DNA substrate containing a 6-base bubble structure with 2 unpaired bases 5' and 3 unpaired bases 3' to the adduct. Formation of this bubble served as a dynamic recognition step in damage processing. UvrB or UvrBC may form one of three stable repair intermediates with DNA substrates, depending upon the state of the DNA surrounding the modified base. The dual incisions were strongly determined by the distance between the adduct and the double-stranded-single-stranded DNA junction of the bubble, and required homologous double-stranded DNA at both incision sites. Remarkably, in the absence of UvrA, UvrBC nuclease can make both 3' and 5' incisions on substrates with bubbles of 3-6 nucleotides, and an uncoupled 5' incision on bubbles of >/=>/=10 nucleotides. These data support the hypothesis that the E.coli and human NER systems recognize and process DNA damage in a highly conserved manner.

Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione S-transferase mu.

Ovarian carcinoma cells 10-fold resistant to the alkylating agent chlorambucil (CBL) were isolated after repeated exposure of the parent cells to gradually escalating concentrations of the drug. The resistant variant, A2780(100), was highly cross-resistant (9-fold) to melphalan and showed lower-level resistance to other cross-linking agents. The resistant A2780(100) cells had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The pi-class GST(s) was the major isoform(s) in both cell lines. However, the resistant A2780(100) cells had at least 11-fold higher GST mu as compared with the parental cells, in which this isoform was barely detectable. A significant induction of GST mu was observed in A2780 cells, but not in the resistant cells, 18 hr after a single exposure to 100 microM CBL. The induction of GST mu by CBL was both time- and concentration-dependent. Assays of the conjugation of CBL with GSH showed that the human mu-class GST had 3.6- and 5.2-fold higher catalytic efficiency relative to the pi- and alpha-class GSTs, respectively. This difference was reflected in the relatively higher (about 6-fold) efficiency of CBL conjugation in A2780(100) cells as compared with the parental cells. These results have demonstrated for the first time a near-linear correlation between CBL resistance and overexpression of mu-class GSTs and suggest that this overexpression maybe responsible, at least in part, for the acquired resistance of ovarian carcinoma cells to CBL, and possibly the other bifunctional alkylating agents. Consistent with this hypothesis, we found evidence for decreased formation of DNA lesions in A2780(100) compared with the drug-sensitive A2780 cells after exposure to CBL.

Hydrophobic forces dominate the thermodynamic characteristics of UvrA-DNA damage interactions.

The Escherichia coli DNA repair proteins UvrA, UvrB and UvrC work together to recognize and incise DNA damage during the process of nucleotide excision repair (NER). To gain an understanding of the damage recognition properties of UvrA, we have used fluorescence spectroscopy to study the thermodynamics of its interaction with a defined DNA substrate containing a benzo[a]pyrene diol epoxide (BPDE) adduct. Oligonucleotides containing a single site-specifically modified N2-guanine (+)-trans-, (-)-trans-, (+)-cis-, or (-)-cis-BPDE adducts were ligated into 50-base-pair DNA fragments. All four stereoisomers of DNA-BPDE adducts show an excitation maximum at 350 nm and an emission maximum around 380 to 385 nm. Binding of UvrA to the BPDE-DNA adducts results in a five to sevenfold fluorescence enhancement. Titration of the BPDE-adducted DNA with UvrA was used to generate binding isotherms. The equilibrium dissociation constants for UvrA binding to (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis- BPDE adduct were: 7.4+/-1.9, 15. 8+/-5.4, 11.3+/-2.7 and 22.4+/-2.0 nM, respectively. There was a large negative change in heat capacity DeltaCpo,obs, (-3.3 kcal mol-1 K-1) accompanied by a relatively unchanged DeltaGoobs with temperature. Furthermore, varying the concentration of KCl showed that the number of ions released upon formation of UvrA-DNA complex is about 3.4, a relatively small value compared to the contact size of UvrA with the substrate. These data suggest that hydrophobic interactions are an important driving force for UvrA binding to BPDE-damaged DNA.

Involvement of molecular chaperonins in nucleotide excision repair. Dnak leads to increased thermal stability of UvrA, catalytic UvrB loading, enhanced repair, and increased UV resistance.

UvrA is one of the key Escherichia coli proteins involved in removing DNA damage during the process of nucleotide excision repair. The relatively low concentrations (nanomolar) of the protein in the normal cells raise the potential questions about its stability in vivo under both normal and stress conditions. In vitro, UvrA at low concentrations is shown to be stabilized to heat inactivation by E. coli molecular chaperones DnaK or the combination of DnaK, DnaJ, and GrpE. These chaperone proteins allow sub-nanomolar concentrations of UvrA to load UvrB through >10 cycles of incision. Guanidine hydrochloride-denatured UvrA was reactivated by DnaK, DnaJ, and GrpE to as much as 50% of the native protein activity. Co-immunoprecipitation assays showed that DnaK bound denatured UvrA in the absence of ATP. UV survival studies of a DnaK-deficient strain indicated an 80-fold increased sensitivity to 100 J/m2 of ultraviolet light (254 nm) as compared with an isogenic wild-type strain. Global repair analysis indicated a reduction in the extent of pyrimidine dimer and 6-4 photoproduct removal in the DnaK-deficient cells. These results suggest that molecular chaperonins participate in nucleotide excision repair by maintaining repair proteins in their properly folded state.

Asbestos increases mammalian AP-endonuclease gene expression, protein levels, and enzyme activity in mesothelial cells.

Only two DNA repair enzymes, DNA polymerase beta and O6-methylguanine-DNA methyltransferase, have been shown to be inducible in mammalian cells by genotoxic agents. We show here that crocidolite asbestos induces the DNA repair enzyme, apurinic/apyrimidinic (AP)-endonuclease, in isolated mesothelial cells, the progenitor cells of malignant mesothelioma. Asbestos at nontoxic concentrations of 1.25 and 2.5 microg/cm2 significantly increased AP-endonuclease mRNA and protein levels as well as enzyme activity (P < 0.05) in a dose-dependent manner in rat pleural mesothelial cells. These increases were persistent from 24 to 72 h after initial exposure to fibers. Changes were not observed with glass beads, a noncarcinogenic particle. Confocal scanning laser microscopy showed that AP-endonuclease was primarily localized in the nucleus but also in mitochondria. Our data are the first to demonstrate the inducibility of AP-endonuclease by a human class I carcinogen associated with oxidant stress in normal cells of the lung.

Patterns of 8-hydroxydeoxyguanosine formation in DNA and indications of oxidative stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos.

Oxidative damage is a proposed mechanism of asbestos-induced carcinogenesis, but the detection of oxidative DNA lesions in target cells of asbestos-induced mesothelioma has not been examined. In studies here, DNA was isolated from both rat pleural mesothelial (RPM) cells and a human mesothelial cell line (MET5A) after exposure in vitro to crocidolite asbestos at various concentrations. DNA was then examined for formation of 8-hydroxydeoxyguanosine (8-OHdG) at 24, 48 and 72 h using HPLC with electrochemical detection. In addition, steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) were assessed as an indication of oxidative stress. Whereas RPM cells showed dose-dependent and significant increases in 8-OHdG formation in response to crocidolite asbestos or iron-chelated crocidolite fibers (but not after exposure to glass beads), MET5A cells showed decreases in 8-OHdG. Both cell types exhibited elevations in message levels of MnSOD. In comparison with human MET5A cells, RPM cells exhibited increased cytotoxicity and apoptosis in response to asbestos, as documented by cell viability assays and flow cytometry analysis using propidium iodide. Results in RPM cells indicate that asbestos causes oxidative damage that may result in potentially mutagenic lesions in DNA and/or apoptosis, despite compensatory increases in expression of an antioxidant enzyme.

Formation of DNA repair intermediates and incision by the ATP-dependent UvrB-UvrC endonuclease.

The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair. To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo[a]pyrene diol epoxide-damaged and structure-specific 50-base pair substrates were constructed. DNA fragments containing a preexisting 3' incision were rapidly and efficiently incised 5' to the adduct. Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision. Experiments with a DNA fragment containing an internal noncomplementary 11-base region surrounding the benzo[a]pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision. In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (Kd approximately 5-10 nM). Formation of this complex was absolutely dependent upon UvrC. Addition to this complex of ATP, but not adenosine 5'-(beta,gamma-iminotriphosphate) or adenosine 5'-(beta, gamma-methylene)triphosphate, caused incision three or four nucleotides 5' to the double strand-single strand junction. The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate. Incision of this Y structure occurs even without DNA damage. Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease.

Interaction of the UvrABC nuclease system with a DNA duplex containing a single stereoisomer of dG-(+)- or dG-(-)-anti-BPDE.

Oligonucleotides containing site-specifically-modified N2-guanine (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-BPDE adducts were ligated into 50-base-pair DNA fragments. These substrates were used in reactions with the Escherichia coli UvrABC nuclease system. The interaction of the UvrA2 and UvrA2B complexes with these four stereoisomers was probed using DNase I footprinting and gel mobility shift assays. DNase I digestion of substrates containing each stereoisomer of BPDE displayed a unique pattern which was consistent with the known structure of these DNA adducts. UvrA and UvrA2B appeared to interact very similarly with all four substrates. Binding of UvrA2 to these substrates produced a 33-bp footprint, and the UvrB--DNA complex resulted in footprint of 24 bp. The UvrABC nuclease system produced bimodal incisions at the eighth phosphate 5' and the fifth, sixth, or seventh phosphate 3' to the modified guanine. The variation of the 3' incision site was linked to the stereochemistry and orientation of the BPDE adduct. For example, the 3' incision of the 50-bp duplex containing (-)-trans-BPDE-N2-guanine was inhibited at the fifth phosphate. UvrABC nuclease incision kinetics revealed a hierarchy of specificity. The intercalative cis isomers were incised more efficiently than the corresponding trans isomers which lie in the minor groove. The (+) enantiomers were incised more efficiently than the (-) form for both cis and trans isomers. These observations reveal that UvrABC nuclease recognition and incision are directly influenced by the conformation of the DNA adduct.

Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal compounds as DNA-protein cross-linking agents: potential suicide DNA lesions.

Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal compounds having the generalized formula M(a)NH2(CH)4NH2M(b) are shown to form specific DNA lesions which can efficiently cross-link proteins to DNA. In this study, the homodinuclear case is represented by M(a) = M(b) = [cis-Pt(Cl2)-(NH3)] and the heterodinuclear case is represented by M(a) = [cis-RuCl2(DMSO)3] and M(b) = [cis-PtCl2(NH3)]. Native and denaturing polyacrylamide gel electrophoresis was used to show the formation of ternary coordination complexes between the metal-treated 49-bp DNA fragment and the Escherichia coli UvrA and UvrB DNA repair proteins. Treatment with proteinase K results in loss of the DNA-protein cross-links. DNA-protein cross-links formed between UvrA and DNA previously modified with the dinuclear metal compounds are reversible with the reducing agent beta-mercaptoethanol. The DNA lesion responsible for efficient DNA-protein cross-linking is most probably a DNA-DNA interstrand cross-link in which each metal atom is coordinated with one strand of the DNA helix. The formation of DNA repair protein associated DNA cross-links, potential "suicide adducts", suggests a novel action mechanism for these anticancer compounds. In addition, these dinuclear metal compounds should be very useful agents for the investigation of a wide range of protein-DNA interactions.