DNA Termini Research Articles

DNA Ligation Catalyzed by Human Topoisomerase IIα

The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion.

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV.

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.

Solution Structure and DNA Binding of the Zinc-finger Domain from DNA Ligase IIIα

DNA ligase IIIα carries out the final ligation step in the base excision repair (BER) and single strand break repair (SSBR) mechanisms of DNA repair. The enzyme recognises single-strand nicks and other damage features in double-stranded DNA, both through the catalytic domain and an N-terminal domain containing a single zinc finger. The latter is homologous to other zinc fingers that recognise damaged DNA, two in the N terminus of poly(adenosine-ribose)polymerase and three in the N terminus of the Arabidopsis thaliana nick-sensing DNA 3′-phosphoesterase. Here, we present the solution structure of the zinc-finger domain of human DNA ligase IIIα, the first structure of a finger from this group. It is related to that of the erythroid transcription factor GATA-1, but has an additional N-terminal β-strand and C-terminal α-helix. Chemical shift mapping using a DNA ligand containing a single-stranded break showed that the DNA-binding surface of the DNA-ligase IIIα zinc finger is substantially different from that of GATA-1, consistent with the fact that the two proteins recognise very different features in the DNA. Likely implications for DNA binding are discussed.

Influence of DNA Structure on DNA Polymerase β Active Site Function: EXTENSION OF MUTAGENIC DNA INTERMEDIATES

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.

Nonhomologous end joining of complementary and noncomplementary DNA termini in mouse testicular extracts

Mammalian somatic cells are known to repair DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) and homologous recombination (HR); however, how male germ cells repair DSBs is not yet characterized. We have previously reported the highly efficient and mostly precise DSB joining ability of mouse testicular germ cell extracts for cohesive and blunt ends, with only a minor fraction undergoing terminal deletion [Mutat. Res. 433 (1999) 1]; however, the precise mechanism of joining was not established. In the present study, we therefore tested the ability of testicular extracts to join noncomplementary ends; we have also sequenced the junctions of both complementary and noncomplementary termini and established the joining mechanisms. While a major proportion of complementary and blunt ends were joined by simple ligation, the small fraction having noncleavable junctions predominantly utilized short stretches of direct repeat homology with limited end processing. For noncomplementary ends, the major mechanism was “blunt-end ligation” subsequent to “fill-in” or “blunting”, with no insertions or large deletions; the microhomology-dependent joining with end deletion was less frequent. This is the first functional study of the NHEJ mechanism in mammalian male germ cell extracts. Our results demonstrate that testicular germ cell extracts promote predominantly accurate NHEJ for cohesive ends and very efficient blunt-end ligation, perhaps to preserve the genomic sequence with minimum possible alteration. Further, we demonstrate the ability of the extracts to catalyze in vitro plasmid homologous recombination, which suggests the existence of both NHEJ and HR pathways in germ cells.

Stable down-regulation of human polynucleotide kinase enhances spontaneous mutation frequency and sensitizes cells to genotoxic agents

Human polynucleotide kinase (hPNK) is a 57.1-kDa monomeric protein with conserved motifs associated with phosphatase and kinase activities. hPNK catalyzes phosphorylation of 5'-DNA termini and dephosphorylation of 3'-DNA termini. Previous studies, employing cell-free systems, have suggested that hPNK participates in the repair of DNA strand breaks. To better define the cellular function of hPNK, a double-stranded small-interfering RNA molecule designed to stably target hPNK transcription was introduced into A549 human lung adenocarcinoma cells. The small-interfering RNA suppressed hPNK gene expression by at least 80-90%. These cells exhibited a 7-fold higher spontaneous mutation frequency based on the development of resistance to ouabain; elevated sensitivity to a broad range of genotoxic agents including gamma-radiation, UVC radiation, methyl methanesulfonate, hydrogen peroxide, and camptothecin; and slower repair of radiation-induced DNA strand breaks. These findings underscore the importance of hPNK in the maintenance of DNA integrity after damage induced by endogenous and exogenous agents.

The Metastasis Suppressor NM23-H1 Possesses 3′-5′ Exonuclease Activity

NM23-H1 belongs to a family of eight gene products in humans that have been implicated in cellular differentiation and development, as well as oncogenesis and tumor metastasis. We have defined NM23-H1 biochemically as a 3'-5' exonuclease by virtue of its ability in stoichiometric amounts to excise single nucleotides in a stepwise manner from the 3' terminus of DNA. The activity is dependent upon the presence of Mg(2+), is most pronounced with single-stranded substrates or mismatched bases at the 3' terminus of double-stranded substrates, and is inhibited by both ATP and the incorporation of cordycepin, a 2'-deoxyadenosine analogue, into the 3'-terminal position. The 3'-5' exonuclease activity was assigned to NM23-H1 by virtue of: 1) precise coelution of enzymatic activity with wild-type and mutant forms of NM23-H1 protein during purification by hydroxylapatite and gel filtration column high performance liquid chromatography and 2) significantly diminished activity exhibited by purified recombinant mutant forms of the proteins. Lysine 12 appears to play an important role in the catalytic mechanism, as evidenced by the significant reduction in 3'-5' exonuclease activity resulting from a Lys(12) to glutamine substitution within the protein. 3'-5' Exonucleases are believed to play an important role in DNA repair, a logical candidate function underlying the putative antimetastatic and oncogenic activities of NM23-H1.

Position-specific Suppression and Enhancement of HIV-1 Integrase Reactions by Minor Groove Benzo[a]pyrene Diol Epoxide Deoxyguanine Adducts: IMPLICATIONS FOR MOLECULAR INTERACTIONS BETWEEN INTEGRASE AND DNA SUBSTRATES

The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication. Integration proceeds in two consecutive integrase-mediated reactions: 3'-processing and strand transfer. To investigate the DNA minor groove interactions of integrase relative to known sites of integrase action, we synthesized oligodeoxynucleotides containing single covalent adducts of known absolute configuration derived from trans-opening of benzo-[a]pyrene 7,8-diol 9,10-epoxide by the exocyclic 2-amino group of deoxyguanosine at specific positions in a duplex sequence corresponding to the terminus of the viral U5 DNA. Because the orientations of the hydrocarbon in the minor groove are known from NMR solution structures of duplex oligonucleotides containing these deoxyguanosine adducts, a detailed analysis of the relationship between the position of minor groove ligands and integrase interactions is possible. Adducts placed in the DNA minor groove two or three nucleotides from the 3'-processing site inhibited both 3'-processing and strand transfer. Inosine substitution showed that the guanine 2-amino group is required for efficient 3'-processing at one of these positions and for efficient strand transfer at the other. Mapping of the integration sites on both strands of the DNA substrates indicated that the adducts both inhibit strand transfer specifically at the minor groove bound sites and enhance integration at sites up to six nucleotides away from the adducts. These experiments demonstrate the importance of position-specific minor groove contacts for both the integrase-mediated 3'-processing and strand transfer reactions.

HMG-D and histone H1 alter the local accessibility of nucleosomal DNA.

There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.

Chemoenzymatic synthesis and antibody detection of DNA glycoconjugates.

A chemoenzymatic approach for the efficient synthesis of DNA-carbohydrate conjugates was developed and applied to an antibody-based strategy for the detection of DNA glycoconjugates. A phosphoramidite derivative of N-acetylglucosamine (GlcNAc) was synthesized and utilized to attach GlcNAc sugars to the 5'-terminus of DNA oligonucleotides by solid-phase DNA synthesis. The resulting GlcNAc-DNA conjugates were used as substrates for glycosyl transferase enzymes to synthesize DNA glycoconjugates. Treatment of GlcNAc-DNA with beta-1,4-galactosyl transferase (GalT) and UDP-Gal produced N-acetyllactosamine-modified DNA (LacNAc-DNA), which could be converted quantitatively to the trisaccharide Lewis X (LeX)-DNA conjugate by alpha-1,3-fucosyltransferase VI (FucT) and GDP-Fuc. The facile enzymatic synthesis of LeX-DNA from GlcNAc-DNA also was accomplished in a one-pot reaction by the combined action of GalT and FucT. The resulting glycoconjugates were characterized by gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and glycosidase digestion experiments. Covalent modification of the 5'-terminus of DNA with carbohydrates did not interfere with the ability of DNA glycoconjugates to hybridize with complementary DNA, as indicated by UV thermal denaturation analysis. The trisaccharide DNA glycoconjugate, LeX-DNA, was detected by a dual DNA hybridization/monoclonal antibody (mAb) detection protocol ("Southwestern"): membrane-immobilized LeX-DNA was visualized by Southern detection with a radiolabeled complementary DNA probe and by Western chemiluminescence detection with a mAb specific for the LeX antigen. The efficient chemoenzymatic synthesis of DNA glycoconjugates and the Southwestern detection protocol may facilitate the application of glycosylated DNA to cellular targeting and DNA glycoconjugate detection strategies.

Which end: dissecting Ku's function at telomeres and double-strand breaks.

The ends of chromosomes are active places. These natural DNA termini must be protected from degradation, recombination, and end-to-end fusions—events that would be ultimately fatal for the genome. In addition, the enzyme telomerase ensures that telomeres in proliferating cells, such as single-celled organisms like budding yeast or the mammalian germ line, are completely replicated. Over the past decade, a plethora of telomereassociated proteins have been identified, based on their ability to regulate telomerase access, provide protection from nucleases and recombinases, and control telomere length (for review, see McEachern et al. 2000; de Lange 2002). In the budding yeast, at least a dozen proteins interact with just the terminal single-stranded overhang, with an additional dozen or more proteins that are associated with the duplex region of the telomere. At mammalian telomeres, which are bound by additional factors that are apparently not present in yeast, the situation is even more complex. To make the task of understanding events that take place at telomeres even more challenging, many telomere-associated proteins perform more than one biochemical activity at chromosome ends. For example, in human cells, the telomere repeat binding factor TRF2 protects chromosomes from end-to-end fusions, and also acts as a negative regulator of telomere length (van Steensel et al. 1998; Loayza and De Lange 2003). The yeast telomere end-binding factor Cdc13 similarly performs several discrete tasks: Cdc13 both positively and negatively regulates telomere elongation by telomerase, as well as shields chromosome termini from unregulated degradation by nucleases (Garvik et al. 1995; Nugent et al. 1996; Booth et al. 2001; Chandra et al. 2001). Analyses of these Cdc13-mediated processes have been greatly aided by separation-of-function mutations that appear to selectively impair each activity. In an analogous manner, recent genetic studies of the catalytic subunit of telomerase have helped to identify discrete regions that mediate enzyme processivity, interaction with other telomerase subunits, or recruitment of the telomerase complex to chromosome termini (Peng et al. 2001; Armbruster et al. 2003; Friedman et al. 2003; Kim et al. 2003). One factor that is critical to several aspects of telomere biology, yet has lagged behind in this genetic dissection, is the Ku heterodimer. Composed of 70and 80-kD subunits, Ku was first appreciated for its role in double-strand break (DSB) repair (Taccioli et al. 1994). In both mammalian and yeast cells, DSBs that arise via developmentally regulated site-specific rearrangements [e.g., V(D)J recombination in vertebrate cells], or are induced experimentally or as a result of environmental assaults such as irradiation, must be readily repaired to maintain genome stability. Ku plays a central role in this process by binding to DNA double-strand ends and recruiting additional factors, which are required to complete repair via nonhomologous end-joining (for review, see Jackson 2002). Somewhat paradoxically, Ku also functions at telomeres, even though these DNA termini are not normally substrates for end-joining reactions. In fact, telomeres must be specifically protected from end-to-end fusions to maintain genomic stability. Furthermore, the role that the Ku heterodimer plays at telomeres is complex, as evidenced by the fact yku70or yku80budding yeast strains, although still viable, exhibit an impressively large number of phenotypes, indicative of severe telomere dysfunction. In these Ku-defective strains, the structure of the extreme single-stranded termini is altered by increased exposure to nucleolytic activity (Gravel et al. 1998; Polotnianka et al. 1998; Maringele and Lydall 2002). Yeast strains that lack Ku also have substantially shortened telomeres (Boulton and Jackson 1996; Porter et al. 1996), in part because of this enhanced nuclease action (A.A. Bertuch and V. Lundblad, in prep.), but primarily because of impaired regulation of telomerase (Peterson et al. 2001; Stellwagen et al. 2003). Additionally, loss of Ku function in yeast disrupts the transcriptional silencing of telomere-proximal genes, by altering the composition of factors that comprise telomeric heterochromatin (Laroche et al. 1998; Nugent et al. 1998; Mishra and Shore 1999). Analysis of yku70or yku80yeast null strains, therefore, has firmly established that the Ku heterodimer is a multifunctional player that contributes to telomere Corresponding author. E-MAIL abertuch@bcm.tmc.edu; FAX (713) 798-3457. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1146603.

Reversible Biotinylation of the 5′‐Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification

The preparation of two reversible biotinylation phosphoramidites and their application in labeling and affinity purification of synthetic oligodeoxyribonucleotides will be described. In both cases, the biotin is linked to the 5'-terminus of DNA through a diisopropyl silyl acetal functionality. This linkage is completely stable under certain postsynthetic cleavage/deprotection conditions, but can be readily broken by fluoride ions, releasing unmodified 5'-OH and 5'-phosphate DNA, respectively. To demonstrate the use of these reversible biotinylation methods, crude DNA was incubated with NeutrAvidin-coated microspheres, full-length biotinylated DNA was efficiently attached to the solid phase, and nonbiotinylated failure sequences and other impurities were readily removed by washing with buffer. Cleavage of the silyl acetal linkage afforded high-quality, full-length, unmodified 5'-OH and 5'-phosphate DNA, respectively, depending on which of the two phosphoramidites was used. It is anticipated that this method will find applications in areas that require efficient isolation of DNA from a complex mixture.

A direct and efficient synthesis method for dumbell-shaped linear DNA using PCR in vitro.

A linear, covalently-closed, dumbbell-shaped DNA vector including a transcription unit is known to have both biological stability and safety and is expected to be useful for gene therapy. We established an easy, quick, and large preparative synthetic method of modified- and unmodified-dumbbell DNA using an intramolecular cyclization at the DNA termini.

Different Genome Maintenance Strategies in Human and Tobacco Cells

In this work, genome maintenance strategies of organisms belonging to different kingdoms (animals versus plants) but of similar genome size were investigated using a novel, universal double-strand break (DSB) repair assay. Different plasmids linearised with KpnI, Acc65I or EcoRV yielding either 3′ or 5′ protruding or blunt DNA termini, respectively, were transfected into HeLa cells and Nicotiana plumbaginifolia protoplasts and assayed for the efficiency and fidelity of DSB repair. We show that the mechanism of break sealing is similar but that drastic differences are seen in the fidelity of repair: in HeLa cells, 50–55% DSBs were repaired precisely, compared to as little as 15–30% in tobacco cells. Moreover, the DSB repair in plants resulted in 30–40% longer deletions and significantly shorter insertions. Combined, these led to more than twofold larger net DNA loss in tobacco cells. Our observations point to possible differences in the strategies of DSB repair and genome maintenance in plants and animals.

Solution structure of a steroid-DNA complex with cholic acid residues sealing the termini of a Watson-Crick duplex.

The three-dimensional structure of a covalent hybrid between cholic acid and the self-complementary DNA hexamer 5'-TGCGCA-3' was solved via two-dimensional NMR and restrained torsion angle molecular dynamics. In the complex, refined to a pairwise rmsd of 0.64 A, the steroid binds to the terminal T:A base pairs via extensive van der Waals contacts but without any hydrogen bonds or detectable dipole-dipole interactions. The contacts involve the methyl groups as well as one edge of the streoid's sterane skeleton and both nucleobases and the deoxyriboses of the terminal base pair of the DNA. The surprising shape complementarity between steroid and the undisturbed DNA termini explains the increase in fidelity and affinity observed for hybridization probes bearing bile acid residues. Since the hydroxyl groups of the steroid do not contribute to the binding of the DNA, they may be derivatized, potentially giving access to a new class of specific binders for blunt ends of Watson-Crick duplexes.

Nucleolytic processing of a protein-bound DNA end by the E. coli SbcCD (MR) complex

SbcCD and other Mre11/Rad50 (MR) complexes are implicated in the metabolism of DNA ends. They cleave ends sealed by hairpin structures and have been postulated to play roles in removing protein bound to DNA termini. Here we provide direct evidence that the Escherichia coli MR complex (SbcCD) removes protein from a protein-bound DNA end by inserting a double-strand break (DSB). These observations indicate a more complex biochemical action than has been assumed previously and argue that this class of protein has the potential to play a direct role in deprotecting protein-bound DNA ends in vivo.

Strategy to Discriminate between High and Low Affinity Bindings of Human Immunodeficiency Virus, Type 1 Integrase to Viral DNA

The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is sequence-specific, and that of the host DNA does not require particular sequence, although in physicochemical studies IN fails to discriminate between the two interactions. Here, such discrimination was allowed thanks to a model system using designed oligonucleotides and peptides as binding structures. Spectroscopic (circular dichroism, NMR, and fluorescence anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly indicated that the amphipathic helix alpha4, located at the catalytic domain surface, is responsible for the specific high affinity binding of the enzyme to viral DNA. Analogues of the alpha4 peptide having increased helicity and still bearing the biologically relevant lysines 156 and 159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are required to achieve high affinity complexes (Kd of 1.5 nm). Data corroborate previous in vivo results obtained with mutated viruses.

Role of Radicals and Singlet Oxygen in Photoactivated DNA Cleavage by the Anticancer Drug Camptothecin: An Electron Paramagnetic Resonance Study

Camptothecin (CPT) is an anticancer drug that inhibits topoisomerase I (Topo I), an enzyme closely linked to cell division, by forming a ternary DNA−CPT−Topo I complex. However, it has been shown that UVA-irradiated CPT in the absence of Topo I produces DNA damage. It has been proposed that free radicals are the key species responsible for the DNA cleavage. It has also been shown that the presence of metal ions enhances the activity of several anticancer drugs. Therefore, we attempt here to explore and identify free radicals generated in these processes. We describe a detailed spectroscopic study of UVA-irradiated CPT and the Cu(II)−CPT complex. From the low-temperature EPR spectrum of the Cu(II)−CPT complex, a proximity between the Cu(II) ion and the 20-hydroxy group of the lactone E ring of CPT is proposed. Upon irradiation (λ = 365 nm) of the Cu(II)−CPT complex in deoxygenated dimethyl sulfoxide (DMSO), the EPR signal of Cu(II) measured in situ at room temperature shows formal first-order exponential decay with a formal half-life of 11 min. By the use of a specific Cu(I) chelating agent, neocuproine, it was shown that, during this process, Cu(II) is reduced to Cu(I). When the photochemical experiments are repeated in oxygen-saturated DMSO solutions, analogous phenomena are observed, characterized by a formal half-life of 16 min for Cu(II), except that there is an induction period of ∼3 min. Application of the spin-trap agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) shows that during this induction period the only radical formed is the superoxide radical, trapped as the •DMPO−O2- adduct. The loss in EPR signal intensity of the Cu(II)−CPT complex upon irradiation is accompanied by the appearance of a new EPR signal at g ≈ 2.0022. Application of the spin traps nitrosodurene (ND) and DMPO revealed that the main radical product formed upon continuous irradiation of CPT in DMSO solutions is the hydroxyl radical (trapped in DMSO as the •CH3 adduct). Application of 2,2,6,6-tetramethyl-4-piperidinol has revealed that irradiation of CPT in aerated DMSO solution also leads to the formation of singlet oxygen (1O2). In the Cu(II)−CPT system, the formation of methyl radicals is suppressed, and the generation of two new radical adducts originating from camptothecin ring cleavage is identified. A mechanism of photochemically generated radicals that include the superoxide radical and the radical cation of the {Cu(II)···CPT•+} complex followed by the reduction of Cu(II) to Cu(I) is proposed. The EPR experiments on irradiated CPT in the absence of copper (II) support the importance of the 20-hydroxy group of the lactone E ring in the antitumor activity of the drug mediated through the initially generated hydroxyl radical. In the presence of Cu(II), there is blockage of the 20-hydroxy group of CPT, and the generation of hydroxyl radicals is strongly suppressed. In this case, there is a mixture of radicals of various origin generated as a result of irradiation, which are capable of causing DNA damage. We propose that the superoxide radical, hydroxyl radical, singlet oxygen, and carbon-centered radicals generated from CPT in close proximity to DNA can cause considerable damage to DNA. These findings would help to explain the experiments indicating that the photoactivated camptothecin interacts specifically with guanines, consistent with preferential stimulation of topoisomerase I cleavage at sites that bear a guanine at their 5‘-DNA terminus. Taken together, our spectroscopic experiments indicate that CPT is a promising photosensitizer and that radicals and singlet oxygen generated upon illumination play a central role in DNA cleavage and in the induction of apoptosis in cancer cells.

Destabilization of the HIV-1 Complementary Sequence of TAR by the Nucleocapsid Protein Through Activation of Conformational Fluctuations

The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two obligatory strand transfer reactions required for the synthesis of a complete proviral DNA by reverse transcriptase. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response region (TAR) sequence at the 3′ end of the genomic RNA and the complementary sequence cTAR at the 3′ terminus of minus strong-stop DNA, the early product of reverse transcription. In order to determine the dynamics of NCp7-mediated nucleic acid destabilization, we investigated by time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly labeled cTAR sequence with NC(12–55) containing NCp7 CCHC zinc fingers and flanking basic amino acid residues. From the chemical rates and the activation energy associated with the conformational fluctuations observed in the absence of NC, it is concluded that such fluctuations are associated with the opening and closing of the double-stranded terminal segments of cTAR. The destabilizing activity of NC(12–55) occurs mainly through a major increase of the opening rate constant of cTAR. Moreover, NC appears to augment the number of pathways between the open and closed states of cTAR, suggesting that it initiates melting of base-pairs at different locations within the terminal segments of cTAR. This activity of NC on the dynamics of cTAR secondary structure is thought to be critical for the formation of the cTAR–TAR complex, which is essential for the specificity and extent of proviral DNA synthesis by reverse transcriptase.

Coordinated Assembly of Ku and p460 Subunits of the DNA-dependent Protein Kinase on DNA Ends is Necessary for XRCC4–ligase IV Recruitment

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA–ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4–ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4–ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4–ligase IV loading to DNA ends since stable DNA-PK/XRCC4–ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.