TRF1 inhibits telomere C-strand DNA synthesis in vitro.

Human TTAGGG repeat-binding factor 1 (TRF1) is involved in the regulation of telomere length in vivo, but the mechanism of regulation remains largely undefined. We have developed an in vitro system for assessing the effect of TRF1 on DNA synthesis using purified proteins and synthetic DNA substrates. Results reveal that TRF1, when bound to telomeric duplex DNA, inhibits DNA synthesis catalyzed by DNA polymerase alpha/primase (pol alpha). Inhibition required that TRF1 be bound to duplex telomeric DNA as no effect of TRF1 was observed on nontelomeric, random DNA substrates. Inhibition was shown to be dependent on TRF1 concentration and the length of the telomeric duplex region of the DNA substrate. When bound in cis to telomeric duplex DNA, TRF1 was also capable of inhibiting pol alpha-catalyzed DNA synthesis on nontelomeric DNA sequences from positions both upstream and downstream of the extending polymerase. Inhibition of DNA synthesis was shown to be specific for TRF1 but not necessarily for the DNA polymerase used in the extension reaction. In a series of control experiments, we assessed T7 DNA polymerase-catalyzed synthesis on a DNA template containing tandem gal4 operators. In these experiments, the addition of the purified Gal4-DNA binding domain (Gal4-DBD) protein has no effect on the ability of T7 polymerase to copy the DNA template. Interestingly, TRF1 inhibition was observed on telomeric DNA substrates using T7 DNA polymerase. These results suggest that TRF1, when bound to duplex telomeric DNA, serves to block extension by DNA polymerases. These results are discussed with respect to the role of TRF1 in telomere length regulation.

Stopped-flow kinetic analysis of replication protein A-binding DNA: damage recognition and affinity for single-stranded DNA reveal differential contributions of k(on) and k(off) rate constants.

Replication protein A (RPA) is a heterotrimeric protein required for many DNA metabolic functions, including replication, recombination, and nucleotide excision repair (NER). We report the pre-steady-state kinetic analysis of RPA-binding DNA substrates using a stopped-flow assay to elucidate the kinetics of DNA damage recognition. The bimolecular association rate, k(on), for RPA binding to duplex DNA substrates is greatest for a 1,3d(GXG), intermediate for a 1,2d(GpG) cisplatin-DNA adduct, and least for an undamaged duplex DNA substrate. RPA displays a decreased k(on) and an increased k(off) for a single-stranded DNA substrate containing a single 1,2d(GpG) cisplatin-DNA adduct compared with an undamaged DNA substrate. The k(on) for RPA-binding single-stranded polypyrimidine sequences appears to be diffusion-limited. There is minimal difference in k(on) for varying length DNA substrates; therefore, the difference in equilibrium binding affinity is mainly attributed to the k(off). The k(on) for a purine-rich 30-base DNA is reduced by a factor of 10 compared with a pyrimidine-rich DNA of identical length. These results provide insight into the mechanism of RPA-DNA binding and are consistent with RPA recognition of DNA-damage playing a critical role in NER.

DNA-Dependent Protein Kinase in Apoptosis.

Programmed cell death or apoptosis can be induced by a variety of mechanisms including genotoxic stress (1-3). The initiation of apoptosis involves the activation of a proteolytic cascade reminiscent of the blood-clotting pathway or activation of pancreatic proteases (4). It has been suggested that a single DNA strand break or persistent DNA adduct is sufficient to induce apoptosis (5). The protease cascade allows for the amplification of the initial signal and results in the degradation of cellular proteins and chromosomal DNA, which are packaged into apoptotic bodies and subsequently removed and recycled by phagocytic cells. The proteases involved in apoptosis employ active site cysteine residues, which catalyze the hydrolysis of the peptide bond following specific aspartic acid residues (6). This class of proteases has been termed caspases for cysteinyl, aspartate-specific proteases. A current view of the caspase cascade is presented in Fig. 1. Genotoxic stress results in the generation of an as yet undefined signal that results in the release of cytochrome C from the intermembrane space of mitochondria into the cytoplasm. It is in the cytoplasm that cytochrome C can form a complex with apocaspase 9, apoptotic protease activating factor-1 (Apaf-1) and deoxyadenosine 5'triphosphate (dATP). This complex is competent for the autoproteolytic activation of caspase-9 (7). Active caspase-9 then cleaves apocaspase-3 to generate an active caspase-3, which is responsible for cleaving specific target proteins, one of which is the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The antiapoptotic factor Bcl-xL can sequester cytochrome C and inhibit the formation of the caspase-9-Apaf-1 complex effectively blocking apoptosis (8). The proapoptotic factor Bcl-xS promotes apoptosis by binding to Bcl-xL and thus blocking the inhibitory effect of this protein (8). Fig. 1. Programmed cell death pathway in response to genotoxic stress.

Cisplatin-DNA adducts inhibit translocation of the Ku subunits of DNA-PK.

We have determined the effect of cisplatin-DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro. The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin-DNA adducts compared to control, undamaged duplex DNA. The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku-DNA complex. In addition to a decrease in DNA-PKcs association, the DNA-PKcs that is bound with Ku at a DNA end containing cisplatin-DNA adducts has a reduced catalytic rate compared to heterotrimeric DNA-PK assembled on undamaged DNA. The position of the cisplatin-DNA lesion from the terminus also effects kinase activation, with maximal inhibition occurring when the lesion is closer to the terminus. These results are consistent with a model for DNA-PK activation where the Ku dimer translocates away from the DNA terminus and facilitates the association of DNA-PKcs which interacts with both Ku and DNA resulting in kinase activation. The presence of cisplatin adducts decreases the ability to translocate away from the terminus and results in the formation of inactive kinase complexes at the DNA terminus. The results are discussed with respect to the ability of cisplatin to sensitize cells to DNA damage induced by ionizing radiation and the ability to repair DNA double-strand breaks.

Physiological functions of protein kinase inhibitors.

Protein kinases are key regulatory enzymes involved in a multitude of biochemical pathways. This chapter will describe the current research on targeting specific protein kinases with inhibitors in attempts to disrupt flux through specific pathways. Targeting specific kinases presents a distinct challenge as there are hundreds of individual kinase enzymes that use ATP as a substrate to phosphorylate specific target molecules. The challenge clearly lies in obtaining specificity for a given kinase, thus allowing inhibition or activation of a specific pathway. This chapter will focus on two areas of kinase inhibitors, those that target the MAP kinase pathway and those directed against the phosphatidylinositol-3 kinase (PI-3K) related kinase family. The cellular and physiological effects of inhibition of the various pathways controlled by these kinases will be reviewed.

Overexpression and purification of human XPA using a baculovirus expression system.

The xeroderma pigmentosum group A protein (XPA) is an essential component of the eukaryotic nucleotide excision repair (NER) process. Recombinant human XPA was expressed in baculovirus-infected insect cells as a [His](6)-tagged fusion protein. A two-column purification procedure resulted in greater than 90% purity for the recombinant protein with a final yield of 0.53 mg from 200 ml of infected cells. The recombinant protein migrated as a doublet of 44 and 42 kDa upon SDS-PAGE consistent with that observed for the native protein. XPA can interact with a number of proteins including replication protein A (RPA) which has been implicated in the initial recognition of damaged DNA. Using a modified ELISA, we demonstrate that the recombinant XPA fusion protein also forms a complex with RPA independent of DNA. The ability of XPA to bind damaged DNA was assessed in an electrophoretic mobility shift assay using globally cisplatin-damaged DNA. The results revealed a slight preference for DNA damaged with cisplatin consistent with its proposed role in the recognition of damaged DNA. The recombinant XPA fusion protein was able to complement cell-free extracts immunodepleted of XPA restoring NER-catalyzed incision of cisplatin-damaged DNA in an in vitro excision repair assay.

Effect of DNA polymerases and high mobility group protein 1 on the carrier ligand specificity for translesion synthesis past platinum-DNA adducts.

Translesion synthesis past Pt-DNA adducts can affect both the cytotoxicity and mutagenicity of the platinum adducts. We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of platinum adducts. The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion replication. In the present study, primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, JM216, or chlorodiethylenetriamine-Pt adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma, and human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) had a similar specificity for translesion synthesis past Pt-DNA adducts (dien >> oxaliplatin >/= cisplatin > JM216). Primer extension assays performed in the presence of high mobility group protein 1 (HMG1), which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216 >> dien). These data were consistent with the results of gel-shift experiments showing similar differences in the affinity of HMG1 for DNA modified with the different platinum adducts. Our studies show that both DNA polymerases and damage-recognition proteins can impart specificity to replicative bypass of Pt-DNA adducts. This information may serve as a model for further studies of translesion synthesis.

Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines.

We have assessed in detail the effect of cisplatin-activated programmed cell death in the cisplatin-sensitive human ovarian cancer cell line A2780 and two drug-resistant subclones, CP70 and C30. To determine whether the differential extent of apoptosis observed between the sensitive and resistant ovarian cancer cell lines was the result of dissimilar upstream signaling events, we assessed the execution of apoptotic events that precede target protein proteolysis and subsequent chromosomal DNA degradation. Proteolytic degradation of procaspase-3 was observed in both the CP70 and C30 cells following IC50 cisplatin treatment, whereas no proteolyzed caspase-3 subunits were detected in the A2780 cells. However, using a direct enzymatic assay measuring cleavage of the synthetic peptide substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide), activity was detected in extracts prepared from A2780 cells treated at the IC90 level of cisplatin and was 2-3-fold less than that of extracts prepared from CP70 and C30 cells. Because the activation of procaspase-3 by caspase-9 requires the release of cytochrome c into the cytoplasm, we determined the level of cytoplasmic cytochrome c in each cell line in response to cisplatin treatment. Consistent with the caspase-3 activation data, a very small increase in cytoplasmic cytochrome c was observed in A2780 cells following cisplatin treatment, whereas dramatic increases were evident in both the CP70 and C30 cell lines. The expression of the mitochondrial factors Bcl-2, Bcl-x, and Bax was determined because each has been implicated in the regulation or release of cytochrome c at the level of the mitochondria. Bcl-2 and Bcl-xL proteins remained relatively unchanged in expression for over 48 h after exposure to cisplatin in the A2780 cell lines. However, within the same time period, expression of Bcl-2 decreased in the CP70- and C30-resistant cell lines, whereas an increase in Bcl-xL expression was observed. Expression of the proapoptotic Bcl-xS protein was observed in only the resistant CP70 and C30 cell lines independent of cisplatin treatment. A change in the expression of Mr 24,000 Bax to a Mr 21,000 isoform was evidenced in the A2780 cells within 48 h of cisplatin treatment and, to a greater extent, in the CP70 and C30 cells, which also expressed a Mr 16,000 Bax variant. Evidence for an alternative apoptotic pathway in A2780 cells was obtained by demonstrating increased FADD expression in response to cisplatin treatment. These results support a model in which cisplatin-induced programmed cell death in the cisplatin-sensitive A2780 and -resistant CP70 and C30 cells proceeds via caspase-3-independent and -dependent pathways, respectively.

Replication protein A (RPA) binding to duplex cisplatin-damaged DNA is mediated through the generation of single-stranded DNA.

Replication protein A (RPA) is a heterotrimeric protein composed of 70-, 34-, and 14-kDa subunits that has been shown to be required for DNA replication, repair, and homologous recombination. We have previously shown preferential binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA compared with the control undamaged DNA (Patrick, S. M., and Turchi, J. J. (1998) Biochemistry 37, 8808-8815). Here we assess the binding of rhRPA to DNA containing site-specific cisplatin-DNA adducts. rhRPA is shown to bind 1.5-2-fold better to a duplex 30-base pair substrate containing a single 1,3d(GpXpG) compared with a 1,2d(GpG) cisplatin-DNA intrastrand adduct, consistent with the difference in thermal stability of DNA containing each adduct. Consistent with these data, a 21-base pair DNA substrate containing a centrally located single interstrand cisplatin cross-link resulted in less binding than to the undamaged control DNA. A series of experiments measuring rhRPA binding and concurrent DNA denaturation revealed that rhRPA binds duplex cisplatin-damaged DNA via the generation of single-stranded DNA. Single-strand DNA binding experiments show that rhRPA binds 3-4-fold better to an undamaged 24-base DNA compared with the same substrate containing a single 1,2d(GpG) cisplatin-DNA adduct. These data are consistent with a low affinity interaction of rhRPA with duplex-damaged DNA followed by the generation of single-stranded DNA and then high affinity binding to the undamaged DNA strand.

Interactions of mammalian proteins with cisplatin-damaged DNA.

We have undertaken the systematic isolation and characterization of mammalian proteins which display an affinity for cisplatin-damaged DNA. Fractionation of human cell extracts has led to the identification of two classes of proteins. The first includes proteins that bind duplex DNA in the absence of cisplatin damage and retain their affinity for DNA in the presence of cisplatin-DNA adducts. The DNA-dependent protein kinase (DNA-PK) falls into this class. The inhibition of DNA-PK phosphorylation activity by cisplatin-damaged DNA has led to the hypothesis that cisplatin sensitization of mammalian cells to ionizing radiation may be mediated by DNA-PK. The second class of proteins identified are those which display a high relative affinity for cisplatin-damaged DNA and a low affinity for undamaged duplex DNA. Proteins that fall into this class include high mobility group 1 protein (HMG-1), replication protein A (RPA) and xeroderma pigmentosum group A protein (XPA). Each protein has been isolated and purified in the lab. The interaction of each protein with cisplatin-damaged DNA has been assessed in electrophoretic mobility shift assays. A series of DNA binding experiments suggests that RPA binds duplex DNA via denaturation and subsequent preferential binding to the undamaged DNA strand of the partial duplex. DNA substrates prepared with photo-reactive base analogs on either the damaged or undamaged DNA strand have also been employed to investigate the mechanism and specific protein-DNA interactions that occur as each protein binds to cisplatin-damaged DNA. Results suggest both damage and strand specificity for RPA and XPA binding cisplatin-damaged DNA.

Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro.

Fractionation of human cell extracts by cisplatin-DNA affinity chromatography was employed to identify proteins capable of binding cisplatin-damaged DNA. A specific protein-DNA complex, termed DRP-3, was identified in an electrophoretic mobility shift assay (EMSA) using a cisplatin-damaged DNA probe. Using this assay we purified DRP-3 and the final fraction contained proteins of 70, 53, 46, 32, and 14 kDa. On the basis of subunit molecular weights, antibody reactivity, and DNA binding activities, DRP-3 was identified as human replication protein A (hRPA). Therefore, we assessed the binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA in vitro. Global treatment of a highly purified completely duplex 44-bp DNA with cisplatin resulted in a 10-20-fold increase in rhRPA binding compared to the undamaged control. The stability of the RPA-DNA complexes was assessed, and NaCl and MgCl2 concentrations that completely inhibited rhRPA binding to undamaged DNA had only a minimal effect on binding to duplex platinated DNA. We assessed rhRPA binding to a duplex DNA containing a single site-specific 1,2-d(GpG) cisplatin adduct, and the results revealed a 4-6-fold increase in binding to this DNA substrate compared to an undamaged control DNA of identical sequence. These results are consistent with RPA being involved in the initial recognition of cisplatin-damaged DNA, possibly mediating DNA repair events. Therefore, we assessed how another cisplatin DNA binding protein, HMG-1, affected the ability of rhRPA to bind damaged DNA. Competition binding assays show minimal dissociation of either protein from cisplatin-damaged DNA during the course of the reaction. Simultaneous addition experiments revealed that HMG-1 binding to cisplatin-damaged DNA was minimally affected by rhRPA, while HMG-1 inhibited the damaged-DNA binding activity of rhRPA. These data are consistent with HMG-1 blocking DNA repair and possibly having the capability to enhance the cytotoxic efficacy of the drug cisplatin.

Mdm-2 phosphorylation by DNA-dependent protein kinase prevents interaction with p53.

In response to genotoxic stress, the p53 tumor suppressor protein exerts a G1 cell cycle arrest that is dependent on its ability to transactivate downstream target genes. This p53-dependent G1 block is reversed by the binding of Mdm-2 to p53, preventing further transactivation. Interestingly, following DNA damage, the mdm-2 gene is also transcriptionally activated by p53, and therefore, the question of how p53 can continue to transactivate genes in the presence of its own negative regulator has remained unanswered. Here, we provide evidence that phosphorylation of Mdm-2 protein by DNA-dependent protein kinase (DNA-PK) blocks its ability to associate with p53 and regulate p53 transactivation. The data support a model by which DNA-PK activation by DNA damage and phosphorylation of Mdm-2 renders the Mdm-2 protein unable to inhibit p53 transactivation, resulting in cell cycle arrest. Following DNA repair, the loss of DNA-PK activity results in newly synthesized Mdm-2 protein that is unphosphorylated and, therefore, capable of binding to p53, allowing cell cycle progression.

Induction of apoptosis in cisplatin-sensitive and -resistant human ovarian cancer cell lines.

In this study, we have assessed the mechanism of cytotoxicity in a series of cisplatin-sensitive and -resistant ovarian carcinoma cells following treatment with equitoxic concentrations of cisplatin. The specific proteolytic degradation and the enzymatic activities of the DNA-dependent protein kinase (DNA-PK) were assessed in the cisplatin-sensitive A2780 cell line and two resistant derivative cell lines, CP70 and C30. Forty-eight h following cisplatin treatment, unattached, apoptotic A2780 cells demonstrated a 20-30% decrease in DNA-PK phosphorylation activity. The resistant CP70 and C30 cell lines showed greater decreases in activity approaching 80 and 90%, respectively. The decreases in kinase activity were attributed to proteolytic degradation of the catalytic subunit of DNA-PK (DNA-PKcs). The extent of degradation mimicked the loss of DNA-PK activity, with the resistant cell lines showing the greatest portion of degraded DNA-PKcs. At the same time point, the ability of the DNA-PK Ku subunits to bind DNA was decreased in apoptotic, unattached cells compared to untreated controls, with the decrease in binding activity being attributed to decreased expression of the Ku subunits. In addition to DNA-PKcs cleavage, specific proteolytic cleavage of the poly(ADP-ribose)polymerase and generation of nucleosome-length DNA ladders was observed in all cell lines following cisplatin treatment. These data suggest that cell death via the accumulation of cisplatin-damaged DNA occurs via apoptosis in both the cisplatin-resistant and -sensitive ovarian cancer cells.

Mechanism of DNA-dependent protein kinase inhibition by cis-diamminedichloroplatinum(II)-damaged DNA.

We have determined the mechanism of DNA-dependent protein kinase (DNA-PK) inhibition by cis-diamminedichloroplatinum(II)-(cisplatin-) damaged DNA. We previously have demonstrated that Ku, the DNA binding subunit of DNA-PK, is capable of binding to DNA duplexes globally damaged with cisplatin but was unable to stimulate DNA-PKcs, the catalytic subunit [Turchi & Henkels (1996) J. Biol. Chem. 271, 2992-3000]. In this report we have assessed Ku binding and DNA-PK stimulation using a series of DNA substrates containing single, site-specific d(GpG), d(ApG), and d(GpXpG) intrastrand cisplatin adducts and a substrate with a single interstrand cisplatin adduct. Results demonstrate that Ku binding is marginally decreased by the presence of cisplatin adducts on each substrate. When assayed for the ability to stimulate DNA-PK, each cisplatin-damaged substrate resulted in significantly decreased activity compared to undamaged DNA controls. The degree of inhibition of both Ku binding and kinase activity varied depending on the specific adduct employed. The inhibition of DNA-PK activity by cisplatin-damaged DNA was observed using either a synthetic peptide or human replication protein A as a substrate. Autophosphorylation of the DNA-PKcs and Ku subunits was also inhibited in reactions performed with cisplatin-damaged DNA, demonstrating that increased autophosphorylation of DNA-PKcs does not account for the decreased kinase activity observed with cisplatin-damaged DNA. Equilibrium binding and initial velocity experiments revealed a less than 2-fold increase in the Kd of Ku and the Km of DNA-PK for DNA containing a single 1,2-d(GpG) cisplatin adduct. The mechanism of DNA-PK inhibition by cisplatin-damaged DNA can be attributed to a large decrease in the Vmax and small increase in Km.

Synthesis of the mammalian telomere lagging strand in vitro.

Using a synthetic telomere DNA template and whole cell extracts, we have identified proteins capable of synthesizing the telomere complementary strand. Synthesis of the complementary strand required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was inhibited by neutralizing antibodies to DNA polymerase alpha. No evidence for RNA-independent synthesis of the lagging strand was observed, suggesting that a stable DNA secondary structure capable of priming the lagging strand is unlikely. Purified DNA polymerase alpha/primase was capable of catalyzing synthesis of the lagging strand with the same requirements as those observed in crude cell extracts. A ladder of products was observed with an interval of six bases, suggesting a unique RNA priming site and site-specific pausing or dissociation of polymerase alpha on the d(TTAGGG)10 template. Removal of the RNA primers was observed upon the addition of purified RNase HI. By varying the input rNTP, the RNA priming site was determined to be opposite the 3' thymidine nucleotide generating a five-base RNA primer with the sequence 5'-AACCC. The addition of UTP did not increase the efficiency of priming and extension, suggesting that the five-base RNA primer is sufficient for extension with dNTPs by DNA polymerase alpha. This represents the first experimental evidence for RNA priming and DNA extension as the mechanism of mammalian telomeric lagging strand replication.

High-mobility group 1 protein inhibits helicase catalyzed displacement of cisplatin-damaged DNA.

We have determined the effect of HMG-1 bound to cisplatin-damaged DNA on the activities of calf helicase E. DNase I protection analysis demonstrated HMG-1 bound a cisplatin-damaged 24 base oligonucleotide annealed to M13mp18. Exonuclease digestion experiments revealed that greater than 90% of the DNA substrates contained a single site specific cisplatin adduct and, maximally, 65% of the substrates were bound by HMG-1. Helicase E catalyzed displacement of the cisplatin-damaged DNA oligonucleotide was inhibited by HMG-1 in a concentration-dependent manner. Time course experiments revealed a decreased rate of displacement in reactions containing HMG-1. The maximum inhibition observed was 55% and taking into account that only 65% of the substrates had HMG-1 bound, approximately 85% inhibition was observed on platinated DNA substrates containing HMG-1. Inhibition of helicase activity was proportional to the amount of substrate bound by HMG-1 based on the displacement and exonuclease assays at varying HMG-1 concentrations. The ability of helicase E to displace an undamaged DNA oligonucleotide from a cisplatin-damaged DNA template was also inhibited by HMG-1. Interestingly, HMG-1 had no effect on the rate of DNA-dependent ATP hydrolysis catalyzed by helicase E on the same DNA substrate. The inhibition of helicase activity by HMG-1 binding cisplatin-damaged DNA further supports a role for HMG-1 inhibiting DNA repair which may contribute to cellular sensitivity to cisplatin.

Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase.

We have identified a series of proteins based on an affinity for cisplatin-damaged DNA. One protein termed DRP-1 has been purified to homogeneity and was isolated as two distinct complexes. The first complex is a heterodimer of 83- and 68-kDa subunits, while the second complex is a heterotrimer of 350-, 83-, and 68-kDa subunits in a 1:1:1 ratio. The 83- and 68-kDa subunits in each complex are identical. The 83-kDa subunit of DRP-1 was identified as the p80 subunit of Ku autoantigen by N-terminal protein sequence analysis and reactivity with a monoclonal antibody directed against human Ku p80 subunit. The 68-kDa subunit of DRP-1 cross-reacted with monoclonal antisera raised against the Ku autoantigen p70 subunit. The 350-kDa subunit was identified as DNA-PKcs, the catalytic subunit of the human DNA-activated protein kinase, DNA-PK. DRP-1/Ku DNA binding was assessed in mobility shift assays and competition binding assays using cisplatin-damaged DNA. Results indicate that DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PKcs. DNA-PK activity was only stimulated with undamaged DNA, despite the ability of Ku to bind to cisplatin-damaged DNA. The lack of DNA-PK stimulation by cisplatin-damaged DNA correlated with the extent of cisplatin-DNA adduct formation. These results demonstrate that Ku can bind cisplatin-damaged DNA but fails to activate DNA-PK. These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA repair processes.

Cisplatin-DNA binding specificity of calf high-mobility group 1 protein.

We have identified a series of proteins with an affinity for cisplatin -damaged DNA using damaged DNA affinity chromatography. We have purified one of these proteins to homogeneity on the basis of a mobility shift assay detecting binding to cisplatin-damaged DNA. The protein was identified as high-mobility group 1 protein (HMG-1) by N-terminal protein sequence analysis. Analysis of a variety of DNA structures revealed that fully duplex DNAs were the best substrates for HMG-1 binding, while partial duplexes were less avidly bound. The decreased levels of binding are attributed to the length of the duplex region of the DNA substrates. A 3-fold increase in binding was observed when a cisplatin-damaged DNA substrate containing a single break in the phosphodiester backbone was joined by DNA ligase. The strict DNA size dependence of binding was also assessed, and a 10-fold increase in binding was observed when the length of the DNA duplex was increased from 44 to 180 base pairs (bp) at the same level of cisplatin damage. HMG-1 binding also was correlated with the degree of cisplatin-DNA damage, suggesting a higher affinity for DNA containing multiple cisplatin adducts. Nuclease degradation of the cisplatin-damaged DNA demonstrated that at the lowest levels of cisplatin damage all of the substrates contained at least one cisplatin adduct. The potential role of HMG-1 in the repair of cisplatin-DNA adducts is discussed.

Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI.

Cleavage specificity of RNase HI was examined on model Okazaki fragments, to determine the likely role of this nuclease in lagging strand DNA replication. Each substrate was prepared by annealing a short RNA primer, made by transcription in vitro, to a single-stranded synthetic DNA template, and subsequently extending the primer by DNA polymerization. The calf thymus RNase HI makes a structure-specific endonucleolytic cleavage in the RNA primer, releasing it intact, and leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. This specific cleavage, one nucleotide upstream of the RNA-DNA junction, is RNA primer sequence- and length-independent. Cleavage specificity is lost if the RNA primer is not extended with DNA, or if the substrate has a nick at the RNA-DNA junction. In addition, the cleavage at a single site requires Mg2+. Cleavage in the presence of Mn2+ is less specific. Neither human immunodeficiency virus reverse transcriptase nor Escherichia coli RNases H perform such a structure-specific cleavage before an RNA-DNA junction. Our work indicates that calf RNase HI is designed to recognize Okazaki fragments. It has the specificity to remove their initiator RNA segments, except for one ribonucleotide, by a single endonucleolytic cleavage in vivo.

Enzymatic completion of mammalian lagging-strand DNA replication.

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis from an upstream fragment to generate a nick, and then ligation. With our substrate, RNase H type I (RNase HI) makes a single cut in the initiator RNA, one nucleotide 5' of the RNA-DNA junction. The double strand specific 5' to 3' exonuclease removes the remaining monoribonucleotide. After dissociation of cleaved RNA, synthesis by DNA polymerase generates a nick, which is then sealed by DNA ligase I. The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in vivo.