DNA Termini Research Articles

Mechanisms of dCMP Transferase Reactions Catalyzed by Mouse Rev1 Protein

The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction. Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus. The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides. Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the V(max) value. Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at a V(max) similar to that for dCMP.

Quantitative differentiation of both free 3' OH and 5' OH DNA ends using terminal transferase-based labeling combined with transmission electron microscopy.

AbstractThe initial concept of apoptosis was proposed by Kerr et al. (1972) (1) and was based on characteristic morphological criteria. Transmission electron microscopy (TEM) allows visualization of fine details of cellular morphology and is one of the most reliable methods for detecting of apoptotic cells. However, TEM has a number of limitations as an apoptosis detection method. First, apoptotic cells detected by TEM are those at the terminal stage of apoptosis. Therefore, TEM cannot identify apoptotic cells entering early stages of apoptotic process. For this purpose, investigators prefer to use fluorescent probes such as a combination of Hoechst 33342 and propidiumiodide (2). Second, chromatin condensation detected by TEM does not necessarily indicate extensive DNA fragmentation. In the thymus, thymocytes with chromatin condensation do not always exhibit extensive nuclear DNA fragmentation (3,4). Recently, Sahara et al. reported the discovery of a caspase-3-activated protein that is required for apoptotic chromatin condensation without DNA fragmentation (5). Therefore, chromatin condensation and DNA fragmentation are independent events of apoptosis. Both agarose gel electrophoresis using extracted DNA and in situ end labeling of DNA strand breaks (ISEL) technique using light microscopy (ISEL/LM) have been widely used for the detecting of DNA fragmentation. To overcome the above-mentioned limitations of TEM, we applied the ISEL technique to TEM (ISEL/TEM technique) (Fig. 1). The procedure of the ISEL/TEM technique is fundamentally the same as that of the ISEL/LM technique, except for the incorporation of immunogold particles, as a substitution for the immunoperoxidase in the ISEL/LM technique (6,7,8). Briefly, dideoxynucleotides labeled with digoxigenin are bound to the 3′ OH DNA ends by reacting with the TdT enzyme in ultrathin sections on a nickel grid. After incubation with an anti-digoxigenin antibody conjugated with 10-nm colloidal gold, the sections are then stained with uranyl acetate and lead citrate and are examined by TEM. The ISEL/TEM technique using immunogold staining has some advantages in terms of detection of apoptotic cells and semiquantification of free 3/t’ OH DNA ends. First, the ISEL/TEM technique enables determination of apoptotic cells entering early stages of apoptosis. It is well known that the increase in the number of sites of newly formed free 3′ OH DNA ends precedes morphological changes associated with apoptosis (6,9). The ISEL/TEM study clearly shows apoptotic cells in the early stage of apoptosis: the moderately increased number of immunogold particles on the initial chromatin condensation but otherwise normal ultrastructural appearance, and in the late stage, the typical chromatin condensation with a numerous immunogold particles in the nuclei (6,8). Second, the ISEL/TEM technique provides information on the mode of DNA cleavage for both apoptosis and necrosis. More than 10 endonucleases have been identified and classified into the following two DNA cleavage types: the 3′ OH/5′ P type and the 3′ P/5′ OH type (8,10). There are only few studies on the mode of DNA cleavage, particularly the 3/t’ P/5/t’ OH type, for both apoptosis and necrosis (8,11,12), although several studies have reported that apoptotic cells possess abundant DNA termini of the 3′ OH/5′ P type (13,14,15). In order to detect the free 5′ OH DNA ends using a nonradioactive method, the 3′ P ends located on the opposite side which hold the 5′ OH ends that were generated by cleavage, were dephosphorylated into 3′ OH ends using alkaline phosphatase (ALP). In this chapter, we describe two techniques for detecting both 3′ OH and 5′ OH ends in a nucleus (see Subheadings 3.3.1, 3.3.2). As described in Subheading 3.3.1, the exact number of 5′ OH ends in a nucleus was determined by calculating the difference between the number of immunogold particles labeled by ISEL/TEM using ALP (ALP/ISEL/TEM) and that by ISEL/TEM, because the ALP/ISEL/TEM technique recognizes both inherent 3′ OH ends and 3′ OH ends newly formed by dephosphorylation. As shown in 3.3.2. (Fig. 1), we can simultaneously detect 3′ OH ends labeled with 5-nm immunogold particles and 3′ P ends labeled with 10-nm immunogold particles in a nucleus, although the number of labeled immunogold particles may decrease due to double staining using different sizes of immunogold particles. Third, the ISEL/TEM technique provides objective data due to the image analysis of quantitative changes for both the 3′ OH and 5′ OH DNA ends in each apoptotic or necrotic cell, when images are processed by employing NIH Image and Adobe Photoshop programs (6,8) (Figs. 2, 3).KeywordsChromatin CondensationEquilibration BufferTransmission Electron Microscopy PhotoCalf Intestinal Alkaline PhosphataseImmunogold ParticleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Involvement of cellular double-stranded DNA break binding proteins in processing of the recombinant adeno-associated virus genome.

Unlike postmitotic tissues in vivo, transduction of cultured cells is poor with recombinant adeno-associated virus (rAAV). The ability of rAAV to transduce cells is greatly enhanced by a variety of agents that induce DNA damage and is elevated in cells defective in the ataxia telangiectasia gene product (ATM), showing increased genomic instability. Here we show that DNA double-stranded break (DSB) repair pathways are involved in the regulation of rAAV transduction efficiency. By quantitative chromatin immunoprecipitation, we found that Ku86 and Rad52 proteins associate with viral DNA inside transduced cells. Both proteins are known to competitively recognize hairpin structures and DNA termini and to promote repair of DSBs, the former by facilitating nonhomologous end joining and the latter by initiating homologous recombination. We found that rAAV transduction is increased in Ku86-defective cells while it is inhibited in Rad52 knockout cells. These results suggest that binding of Rad52 to the rAAV genome might be involved in processing of the vector genome through a homologous recombination pathway.

Assembly and catalysis of concerted two-end integration events by Moloney murine leukemia virus integrase.

Retroviral integration results in the stable and coordinated insertion of the two termini of the linear viral DNA into the host genome. An in vitro concerted two-end integration reaction catalyzed by the Moloney murine leukemia virus (M-MuLV) integrase (IN) was used to investigate the binding and coordination of the two viral DNA ends. Comparison of the two-end integration and strand transfer assays indicates that zinc is required for efficient concerted integration utilizing plasmid DNA as target. Complementation assays using a pair of nonoverlapping integrase domains, consisting of the HHCC domain and the core/C-terminal region, yielded products containing the correct 4-base target site duplication. The efficiency of the coordinated two-end integration varied depending on the order of addition of the individual protein and DNA components in the complementation assay. Two-end integration was most efficient when the long terminal repeat (LTR) was premixed with either the target DNA or the HHCC domain. The preference for two-end integration through preincubation of the HHCC finger with the viral DNA supports the role of this domain in the recognition and/or positioning of the LTR.

Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing.

Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

Production, characterization, and epitope mapping of monoclonal antibodies against human polydeoxyribonucleotide kinase.

Polydeoxyribonucleotide kinase (PNK) is a mammalian DNA repair enzyme that has the capacity to phosphorylate 5' DNA termini and dephosphorylate 3' DNA termini. A series of murine monoclonal antibodies (MAbs) was raised against the full-length recombinant human PNK. Seven of these antibodies were selected and characterized by enzyme immunoassay, Western blot analysis, and their capacity to immunoprecipitate PNK. The epitope location was defined by cyanogen bromide digestion and by using a truncated PNK for Western blot analysis. All of the MAbs recognize a single 60-kDa protein in human cell extracts. PNKs from calf, monkey, and Chinese hamster cell and tissue extracts were also detected by some or all of the MAbs. These antibodies can be successfully used for the cellular, biochemical, and functional analysis of PNK in different mammalian cell lines.

G-overhang dynamics at Tetrahymena telomeres

To learn more about the structure of the DNA terminus at Tetrahymena thermophila telomeres, we have devised a ligation-mediated primer extension protocol to accurately measure the length of the G-strand overhang. We show that overhang length and the identity of the 3'-terminal nucleotide are tightly regulated. The majority of overhangs terminate in the sequence 5'-TTGGGGT and >80% are either 14-15 or 20-21 nucleotides in length. No significant changes in overhang length were detected as cells traversed the cell cycle. However, changes in length distribution were observed when cells exited the cell cycle, indicating an altered balance between DNA synthesis and degradation or end protection. We also provide evidence that rDNA molecules have overhangs on both telomeres. Full-length rDNA could be cloned by a strategy that depends on overhangs being present at both ends. Moreover, analysis of leading strand telomeres revealed that a significant fraction have overhangs > or =5 nucleotides. Our results indicate that generation of the terminal telomeric DNA structure is highly regulated and requires several distinct DNA-processing events.

Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines.

The heterodimeric Ku protein (composed of the Ku 86 and Ku 70 sub-units) is a nuclear protein which binds to DNA termini without sequence specificity. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex that is required for the repair of DNA double-strand breaks in mammalian cells. We studied the expression and function of Ku/DNA-PK during granulocytic differentiation of two human promyelocytic cell lines, HL60 and NB4, a process associated to decreased radiation resistance. After 3 days exposure to differentiating agents (either all-trans-retinoic acid or DMSO), Ku binding to double stranded (ds)-DNA ends declined dramatically whereas Ku protein levels remain unchanged. The nuclear, but not cytoplasmic, fraction of differentiated HL60 cells extracts exhibited a heat-sensitive inhibitory activity towards DNA binding of recombinant Ku heterodimer. We further demonstrate that immunoprecipitation of Ku is impaired in extracts from differentiated cells by using two antibodies that recognize epitopes within the C-terminus DNA binding domains of Ku 70 and Ku 86 proteins. These results favor the hypothesis of a protein interacting with Ku that would prevent DNA binding of heterodimerized Ku protein by steric hindrance.

Domain Structure and Mutational Analysis of T4 Polynucleotide Kinase

T4 polynucleotide kinase (Pnk) is the founding member of a family of 5'-kinase/3'-phosphatase enzymes that heal broken termini in RNA or DNA by converting 3'-PO(4)/5'-OH ends into 3'-OH/5'-PO(4) ends, which are then suitable for sealing by RNA or DNA ligases. Here we employed site-directed mutagenesis and biochemical methods to dissect the domain structure of the homotetrameric T4 Pnk protein and to localize essential constituents of the apparently separate active sites for the 5'-kinase and 3'-phosphatase activities. We characterized deletion mutants Pnk(42-301) and Pnk(1-181), which correspond to domains defined by proteolysis with chymotrypsin. Pnk(1-181) is a monomer with no 3'-phosphatase and low residual 5'-kinase activity. Pnk(42-301) is a dimer with no 5'-kinase and low residual 3'-phosphatase activity. Four classes of missense mutational effects were observed. (i) Mutations K15A, S16A, and D35A inactivated the 5'-kinase but did not affect the 3'-phosphatase or the tetrameric quaternary structure of T4 Pnk. 5'-kinase activity was ablated by the conservative mutations K15R, K15Q, and D35N; however, kinase activity was restored by the S16T change. (ii) Mutation D167A inactivated the 3'-phosphatase without affecting the 5'-kinase or tetramerization. (iii) Mutation D85A caused a severe decrement in 5'-kinase activity and only a modest effect on the 3'-phosphatase; the nearby N87A mutation resulted in a significantly reduced 3'-phosphatase activity and slightly reduced 5'-kinase activity. D85A and N87A both affected the quaternary structure, resulting in a mixed population of tetramer and dimer species. (iv) Alanine mutations at 11 other conserved positions had no significant effect on either 5'-kinase or 3'-phosphatase activity.

Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces.

Linear plasmids and chromosomes of the bacterial genus Streptomyces have proteins of unknown characteristics and function linked covalently to their 5' DNA termini. We purified protein attached to the end of the pSLA2 linear plasmid of Streptomyces rochei, determined the N-terminal amino acid sequence, and used this information to clone corresponding genes from a S. rochei cosmid library. Three separate terminal protein genes (here designated as tpgR1, tpgR2, and tpgR3), which map to the S. rochei chromosome and to 100-kb and 206-kb linear plasmids contained in S. rochei, were isolated and found to encode a family of similar but distinct 21-kD proteins. Using tpgR1 to probe a genomic DNA library of Streptomyces lividans ZX7, whose linear chromosome can undergo transition to a circular form, we isolated a S. lividans chromosomal gene (tpgL) that we found specifies a protein closely related to, and functionally interchangeable with, TpgR proteins for pSLA2 maintenance in S. lividans. Mutation of tpgL precluded propagation of the pSLA2 plasmid in a linear form and also prevented propagation of S. lividans cells that contain linear, but not circular, chromosomes, indicating a specific and essential role for tpg genes in linear DNA replication. Surprisingly, Tpg proteins were observed to contain a reverse transcriptase-like domain rather than sequences in common with proteins that attach covalently to the termini of linear DNA replicons.

Accurate in Vitro End Joining of a DNA Double Strand Break with Partially Cohesive 3′-Overhangs and 3′-Phosphoglycolate Termini: EFFECT OF Ku ON REPAIR FIDELITY

To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3'-overhangs and a one-base gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3' --> 5' resection before ligation, was formed. Similar results were seen for a substrate with 5'-overhangs and recessed 3'-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation.

Role of DNA-dependent protein kinase in recognition of radiation-induced DNA damage in human peripheral blood mononuclear cells.

The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in radiation-induced DNA damage recognition. The complex includes the ku heterodimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKCS: The activation of the DNA-PK complex was studied in X-irradiated peripheral blood mononuclear cells (PBMC) from subjects of different ages. Radiation-induced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were determined in nuclear and cytoplasmic extracts. DNA-binding activity was increased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extracts regardless of age. The radiation-induced activation of the DNA-PK complex may result from the increased concentrations of ku 80 and DNA-PKcs in the cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks. These findings suggest major steps involved in DNA-PK activation, and the intracellular and molecular changes that may account for the age-dependent impairment of DNA repair capacity in irradiated mammalian cells.

Positioning the 3′-DNA Terminus for Topoisomerase II-mediated Religation

Despite the importance of the topoisomerase II DNA cleavage/rejoining cycle to genomic integrity, the mechanistic details of religation are poorly understood. Topoisomerase II utilizes covalent protein-DNA interactions to align the 5'-termini of cleaved DNA for religation. However, because the enzyme does not form covalent bonds with the 3'-DNA termini, the basis for the alignment of the 3'-ends is less clear. Three major possibilities exist. The 3'-termini may be positioned for religation (i) by base pairing to their complementary DNA strands, (ii) by base stacking to the adjacent residues, or (iii) by noncovalent interactions with topoisomerase II. To distinguish between these possibilities, the ability of human topoisomerase IIalpha to religate a series of oligonucleotides with altered base pairing or base stacking at their 3'-termini was determined. Substrates containing modifications that disrupted terminal base pairing or base stacking with-out affecting the 3'-terminal base were resealed at wild-type rates. In contrast, substrates that lacked the terminal base (or contained an altered base) displayed very low rates of religation. On the basis of these results, we propose that topoisomerase II positions the 3'-DNA termini for religation through noncovalent protein-DNA contacts.

NucA is required for DNA cleavage during transformation of Bacillus subtilis.

We have re-examined the roles of nucA and nin, in the transformation of Bacillus subtilis as conflicting accounts have been presented concerning the importance of these genes for transformation. The present report demonstrates that nucA deficiency lowers the rate of DNA transport and that NucA is needed for the double-strand cleavage of transforming DNA, probably acting directly as an endonuclease. A relative paucity of DNA termini, resulting from the absence of this endonuclease activity, most probably accounts for the decreased transport rate. NucA is a bitopic integral membrane protein, with its C-terminus external to the membrane where it is appropriately located to effect the cleavage of bound transforming DNA. We have also investigated the roles of the known competence genes in the DNA processing that accompanies transformation in B. subtilis. The genes that are required for DNA transport (comEA, comEC and comFA) are also required for the degradation of the non-transforming strand that accompanies internalization, but comEC and comFA are not needed for the double-strand cleavage that occurs external to the cell membrane.

DNase protection analysis of retrovirus integrase at the viral DNA ends for full-site integration in vitro.

Retrovirus intasomes purified from virus-infected cells contain the linear viral DNA genome and integrase (IN). Intasomes are capable of integrating the DNA termini in a concerted fashion into exogenous target DNA (full site), mimicking integration in vivo. Molecular insights into the organization of avian myeloblastosis virus IN at the viral DNA ends were gained by reconstituting nucleoprotein complexes possessing intasome characteristics. Assembly of IN-4.5-kbp donor complexes capable of efficient full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. DNase I footprint analysis of assembled IN-donor complexes capable of full-site integration shows that wild-type U3 and other donors containing gain-of-function attachment site sequences are specifically protected by IN at low concentrations (<20 nM) with a defined outer boundary mapping ~20 nucleotides from the ends. A donor containing mutations in the attachment site simultaneously eliminated full-site integration and DNase I protection by IN. Coupling of wild-type U5 ends with wild-type U3 ends for full-site integration shows binding by IN at low concentrations probably occurs only at the very terminal nucleotides (<10 bp) on U5. The results suggest that assembly requires a defined number of avian IN subunits at each viral DNA end. Among several possibilities, IN may bind asymmetrically to the U3 and U5 ends for full-site integration in vitro.

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine: a novel anticancer nucleoside analog that causes both DNA strand breaks and G(2) arrest.

The mechanism of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) action was investigated in human lymphoblastoid CEM cells and myeloblastic leukemia ML-1 cells. CNDAC was metabolized to its 5'-triphosphate and incorporated into DNA, which was associated with inhibition of DNA synthesis. After incubation of cells with [(3)H]CNDAC, metabolites were detected in 3'-->5' phosphodiester linkage and at the 3' terminus of cellular DNA. Specific enzymatic hydrolysis of DNA demonstrated that the parent nucleoside and its 2'-epimer 2'-C-cyano-2'-deoxy-2-ribo-pentofuranosylcytosine accounted for approximately 65% of the total analogs incorporated into DNA and essentially all of the drug in the 3'-->5' phosphodiester linkage. In contrast, all detectable radioactivity at 3' termini was associated with 2'-C-cyano-2',3'-didehydro-2',3'-dideoxycytidine. This de facto DNA chain-terminating nucleotide arises from an electronic characteristic and cleavage of the 3'-phosphodiester bond subsequent to the addition of a nucleotide to the incorporated CNDAC moiety by beta-elimination, a process that generates a single strand break in DNA. Investigation of the biological consequences of these actions indicated that, after incubation with cytostatic concentrations of CNDAC, cell cycle progression was delayed during S phase, but that cells arrested predominantly in the G(2) phase. This differed from the S phase-arresting actions of ara-C and gemcitabine, other deoxycytidine analogs that inhibit DNA replication but do not cause strand breaks. Thus, once incorporated into DNA, the CNDAC molecule appears to act by a dual mechanism that 1) delays the progress of further DNA replication, but 2) upon addition of a deoxynucleotide results in the conversion of the incorporated analog to a de facto DNA chain terminator at the 3' terminus of a single strand break. It is likely that DNA strand breaks trigger cell cycle arrest in G(2).

Inhibition of HIV-1 integrase by modified oligonucleotides derived from U5' LTR.

Retroviral integrase catalyzes integration of double-stranded viral DNA into the host chromosome by a process that has become an attractive target for drug design. In the 3' processing reaction, two nucleotides are specifically cleaved from both 3' ends of viral DNA yielding a 5' phosphorylated dimer (pGT). The resulting recessed 3' hydroxy groups of adenosine provide the attachment sites to the host DNA in the strand transfer reaction. Here, we studied the effect of modified double-stranded oligonucleotides mimicking both the unprocessed (21-mer oligonucleotides) and 3' processed (19-mer oligonucleotides) U5 termini of proviral DNA on activities of HIV-1 integrase in vitro. The inhibitions of 3' processing and strand transfer reactions were studied using 21-mer oligonucleotides containing isopolar, nonisosteric, both conformationally flexible and restricted phosphonate internucleotide linkages between the conservative AG of the sequence CAGT, and using a 21-mer oligonucleotide containing 2'-fluoroarabinofuranosyladenine. All modified 21-mer oligonucleotides competitively inhibited both reactions mediated by HIV-1 integrase with nanomolar IC50 values. Our studies with 19-mer oligonucleotides showed that modifications of the 3' hydroxyl significantly reduced the strand transfer reaction. The inhibition of integrase with 19-mer oligonucleotides terminated by (S)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine, 9-(2-phosphonomethoxyethyl)adenine, and adenosine showed that proper orientation of the 3' OH group and the presence of the furanose ring of adenosine significantly influence the strand transfer reaction.

Interactions of the Human T-cell Leukemia Virus Type-II Integrase with the Conserved CA in the Retroviral Long Terminal Repeat End

Retroviral integrases (INs) interact with termini of retroviral DNA in the conserved 5'-C(A/G)T. For most integrases, modifications of critical moieties in the major and minor grooves of these sequences decrease 3'-processing. However, for human immunodeficiency virus type-2 (HTLV-2) IN, the replacement of the guanine with 6-methylguanine or hypoxanthine not only reduced 3'-processing, but also promoted cleavage at a second site. This novel cleavage activity required an upstream ACA, unique to the HTLV-2 U5 end. 3'-Processing assays with additional isosteric modifications at Gua and filter binding experiments revealed that the mechanism of the second site cleavage differed among the major groove, minor groove, and mismatch modifications. Importantly, the decrease in 3'-processing activity noted with the minor groove and mismatch modifications were attributed to a decrease in binding. Major groove modifications, however, decreased the level of 3'-processing, but did not affect binding. This suggests that integrase binds the viral end through the minor groove, but relies on major groove contacts for 3'-processing. Several modifications were also examined in strand transfer and disintegration substrates. HTLV-2 IN showed reduced activity with strand transfer and disintegration substrates containing major groove, but not minor groove modifications. This suggests major groove interactions at guanine also provide an important role in these reactions.

Evaluation of retroviral ribonuclease H activity.

Reverse transcription is the process whereby the single-stranded RNA genome of a retrovirus is converted into double-stranded DNA by the DNA polymerase and ribonuclease H (RNase H) activities of virus-coded reverse transcriptase (RT) (,). The former activity of reverse transcriptase has been studied extensively, both as a potential target for antiviral therapy and a tool for molecular biology. However, increasing attention has been directed recently toward understanding the roles of RNase H activity, both nonspecific and highly specialized, in reverse transcription (reviewed in ref. ). A complete summary of the process is depicted in Fig. 1. Quantitatively, the most important RNase H function is to degrade the RNA strand of ∼10,000 bp RNA-DNA replication intermediate, eliminating the need for strand-displacement during synthesis of the second or (+) strand. This degradation is largely nonspecific, yet several cleavage events demand a high degree of precision, such as scission of the RNA strand at specific positions near the 3− termini of small, purine-rich stretches of RNA called polypurine tracts (PPTs). These RNAs serve as primers for (+) strand synthesis, but must also be removed by the enzyme once initiation has occurred. The (−;) strand primer, a host-derived tRNA, is removed after its 3′-terminus is copied during (+) strand synthesis. If the (+) and (−) strand primers are not precisely removed, the resulting errors would alter the 5′- and 3′-termini of viral DNA (Fig. 1 E). This would inhibit integration of viral DNA into the host-cell genome and perhaps adversely affect the viability of the virus at later stages of its life cycle.

XRCC1 Stimulates Human Polynucleotide Kinase Activity at Damaged DNA Termini and Accelerates DNA Single-Strand Break Repair

XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-β and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.