The Bloom's syndrome gene product interacts with topoisomerase III.

Bloom's syndrome is a rare genetic disorder associated with loss of genomic integrity and a large increase in the incidence of many types of cancer at an early age. The Bloom's syndrome gene product, BLM, belongs to the RecQ family of DNA helicases, which also includes the human Werner's and Rothmund-Thomson syndrome gene products and the Sgs1 protein of Saccharomyces cerevisiae. This family shows strong evolutionary conservation of protein structure and function. Previous studies have shown that Sgs1p interacts both physically and genetically with topoisomerase III. Here, we have investigated whether this interaction has been conserved in human cells. We show that BLM and hTOPO IIIalpha, one of two human topoisomerase III homologues, co-localize in the nucleus of human cells and can be co-immunoprecipitated from human cell extracts. Moreover, the purified BLM and hTOPO IIIalpha proteins are able to bind specifically to each other in vitro, indicating that the interaction is direct. We have mapped two independent domains on BLM that are important for mediating the interaction with hTOPO IIIalpha. Furthermore, through characterizing a genetic interaction between BLM and TOP3 in S. cerevisiae, we have identified a functional role for the hTOPO IIIalpha interaction domains in BLM.

Purification and characterization of human DNA topoisomerase IIIalpha.

Human topoisomerase IIIalpha (hTopo IIIalpha), the recently identified first member of the topoisomerase IA subfamily in humans, has a central domain which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In order to determine the properties of hTopo IIIalpha, compared to those of other topoisomerase IA subfamily members, we purified this enzyme to near homogeneity, together with an active site-mutant Y337F. We show that hTopo IIIalpha is able to relax negatively supercoiled DNA in a distributive manner, leading to the total disappearance of the initial substrate and the appearance of intermediate topoisomers. This DNA relaxation activity is magnesium-dependent, although a low concentration of MgCl2is sufficient to obtain efficient catalysis. 32P-transfer experiments demonstrated that hTopo IIIalpha is able to cleave a single-stranded oligonucleotide and to bind covalently to the 5'-end of the cleaved DNA. Addition of 0.5 M NaCl reverses the reaction, leading to the religation of the oligo-nucleotide. Experiments utilizing several different single-stranded oligonucleotides permitted us to map several cleavage sites and to deduce a consensus sequence for DNA cleavage (CANNN downward arrow), which is different from that for other members of the Topo IA subfamily.

Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein.

We tested whether the selection of target sites can be manipulated by fusing retroviral integrase with a sequence-specific DNA-binding protein. A hybrid protein that has the Escherichia coli LexA protein fused to the C terminus of the human immunodeficiency virus type 1 integrase was constructed. The fusion protein, IN1-288/LA, retained the catalytic activities in vitro of the wild-type human immunodeficiency virus type 1 integrase (WT IN). Using an in vitro integration assay that included multiple DNA fragment as the target DNA, we found that IN1-288/LA preferentially integrated viral DNA into the fragment containing a DNA sequence specifically bound by LexA protein. No bias was observed when the LexA-binding sequence was absent, when the fusion protein was replaced by WT IN, or when LexA protein was added in the reaction containing IN1-288/LA. A majority of the integration events mediated by IN1-288/LA occurred within 30 bp of DNA flanking the LexA-binding sequence. The specificity toward the LexA-binding sequence and the distribution and frequency of target site usage were unchanged when the integrase component of the fusion protein was replaced with a variant containing a truncation at the N or C terminus or both, suggesting that the domain involved in target site selection resides in the central core region of integrase. The integration bias observed with the integrase-LexA hybrid shows that one effective means of altering the selection of DNA sites for integration is by fusing integrase to a sequence-specific DNA-binding protein.

Impairment of Moloney murine leukemia virus integration in a cell line underexpressing DNA topoisomerase II.

The possible intervention of nuclear proteins as cofactors of integrase-catalyzed integration of retroviral DNA into the host cell genome is not fully understood. Among various nuclear proteins, DNA topoisomerase II appears to be a plausible candidate. This hypothesis is supported by a series of evidence, including the fact that integration is markedly affected by the topology of the target DNA and mainly occurs in transcribed regions in which topoisomerase II is preferentially located. In an attempt to confirm the validity of this hypothesis, we have comparatively investigated the early stages of a recombinant Moloney murine leukemia virus (psi neo) in two related Chinese hamster cell lines (DC3F and R/DC3F) expressing different levels of both isoforms of topoisomerase II. R/DC3F is derived from the parental cell line DC3F and displays a resistant phenotype towards the usual anticancer topoisomerase II inhibitors (actinomycin D, doxorubicin, and taxol). Results show that the early stages of the retroviral cycle are markedly impaired in cells underexpressing topoisomerase II (R/DC3F). This alteration mimics Fv-1 restriction and is characterized by about a 6-fold decrease in viral DNA synthesis and total inhibition of viral genome integration. The specific impairment of integration in R/DC3F cells compared to DC3F cells is assessed by the absence of G418-resistant colonies upon viral infection and a lack of the viral genome in cellular nuclear DNA as detected by the PCR procedure. These features are observed in relevant infecting conditions leading, in both cell lines, to the same amount of linear viral DNA and to the occurrence of two long terminal repeats containing circular DNA in the nuclear fractions.

Triplex-mediated inhibition of HIV DNA integration in vitro.

Integration of human immunodeficiency virus (HIV) DNA into the genome of host cells is an obligatory step in the replicative cycle of the virus. The overall process is carried out in vitro by a single viral protein, the integrase, which binds to short sequences located at the ends of viral DNA long terminal repeats (LTRs). These end sequences are highly conserved in all HIV genomes and are therefore attractive targets for selective DNA binding compounds. The integrase-binding site located in U3 LTR contains a purine motif, 5'-GGAAGGG-3' which can be selectively targeted by oligonucleotide-intercalator conjugates. Under neutral pH and physiological temperature, these conjugates readily form a stable complex with the viral DNA which involves a short DNA triplex. Triple-helix formation prevents the catalytic functions of the integrase in vitro which results in a sequence-specific inhibition of the U3 integration process.

Potentiation of 2',3'-dideoxycytidine (ddC) by hydroxyurea and thymidine on the Moloney murine leukemia virus (MoMLV) early replicative steps.

Combinations of ddC with either the ribonucleotide reductase inhibitor hydroxyurea (HU) or with the natural nucleoside thymidine have been investigated on the cycle of a defective (psi neo) Moloney Leukemia Virus (MoMLV) using 3T3 fibroblasts as host cells. In this experimental model, ddC displayed very poor antiviral action which was obvious given an IC50 value close to 100 microM, i.e. an efficiency about thirty thousand fold lower than that of AZT. Both HU and thymidine alone resulted in significant inhibition of MoMLV replication with IC50 values of 40 microM and 100 microM respectively. The combination of ddC with 50 microM HU lowered the IC50 of ddC by a factor of 10. A similar but more pronounced effect was obtained by combining ddC and 100 microM thymidine, which decreases the IC50 value of ddC by a factor of 50. Combining 40 microM ddC and 100 microM thymidine resulted in the quite complete inhibition of viral replication. These results show that in cell types with strongly restricted ddC action, combination treatment with compounds known to ultimately decrease dCTP biosynthesis leads to the restoration of efficient antiviral activity.

Inhibition of the in vitro integration of Moloney murine leukemia virus DNA by the DNA minor groove binder netropsin.

In search of potential inhibitors of integration of retroviral DNA into host cells genome, we have investigated the effect of the external DNA binder netropsin on the in vitro insertion of long terminal repeat (LTR) ends of Moloney murine leukemia virus (M.MuLV) as catalysed by integrase purified from baculovirus strain expression vector. In agreement with the preferential binding of netropsin to A+T rich sequences, footprinting experiments have shown that this drug selectively binds to the 5'-TTTCAT LTR end sequence which is included in the DNA binding site of integrase. This feature results in the potent inhibition of both reactions involved in the insertion process, namely, nucleolytic cleavage and strand transfer. The relation between netropsin binding to A+T rich region of M.MuLV LTR end and inhibition of insertion is strongly suggested from the inability of the drug to inhibit the insertion of HIV U3 LTR end which displays a G+C rich sequence. Selective inhibition of integration of viral DNA appears to be feasible using drugs recognizing LTR end sequences.

Exogenous nucleosides promote the completion of MoMLV DNA synthesis in G0-arrested Balb c/3T3 fibroblasts.

We studied Moloney murine leukemia virus replication in newly infected Balb c/3T3 cells brought to the G0 phase by serum depletion. Using the polymerase chain reaction method, we showed that Moloney murine leukemia virus can be efficiently internalized in nonproliferating fibroblasts, although reverse transcription of the viral RNA in these cells remains incomplete. It seems likely that a lower availability of deoxyribonucleotides in G0-arrested cells is responsible for this premature termination of the reverse transcription step. Accordingly, the addition of high concentrations of nucleosides to the culture medium of nondividing cells simultaneously with infection enables them to complete the reverse transcription process, without re-initiating the cell cycle. Inhibition of reverse transcription by hydroxyurea confirms the dependence of this retroviral step on the intracellular nucleotide pool rather than on the precise arrest point of the host cell cycle. Furthermore, the pyrimidine nucleotide pool, and more particularly the cytidine pool, appears to play a central regulatory role in this step.

Triple helix formation with short oligonucleotide-intercalator conjugates matching the HIV-1 U3 LTR end sequence.

In an attempt to target short purine sequences in view of pharmacological application, we have synthesized three new TFO (triple-helix-forming oligonucleotide) conjugates in which an intercalating oxazolopyridocarbazole (OPC) chromophore is linked by a pentamethylene linker to a 7-mer oligonucleotide matching the polypurine/polypyrimidine sequence located in the HIV-1 U3 LTR end region. The TFO moiety of conjugates are 5'CCTTCCC, 5'GGGAAGG, and 5'GGGTTGG. Their ability to bind to double-stranded DNA targets was examined. This binding is demonstrated by a footprinting technique using DNase I as a cleaving agent. The complex involved intermolecular pyr-pur*pyr or pur-pur*pyr triple helix. Pyrimidine TFO-OPC binds in a pH-dependent manner, whereas the others do not. The formation of the complex has been investigated at neutral pH and increasing temperature. We observed that the protection due to the purine and mixed TFO-OPC was pH independent and remained identical up to 40 degrees C. To determine the position of the OPC chromophore, molecular modeling was undertaken on the purine-conjugate/target complex. It has been suggested that the complex involved the intercalation of the OPC at the triplex-duplex junction with a small unwinding at the next excluded site.

Selective binding to polynucleotides of the hybrid intercalating groove binder bis(pyrrolecarboxamide)-oxazolopyridocarbazole: a molecular modeling study.

In order to further characterize the binding of the hybrid molecule NetOPC [bis(pyrrolecarboxamide)-oxazolopyridocarbazole conjugate] to double-stranded DNA, we have performed a molecular modeling study to investigate the binding modes of the complexes possibly formed between NetOPC and synthetic polynucleotides poly [(dA-dT)]2, poly [(dA).d(T)], and poly [d(G-C)]2 and interpreted the results in the light of the experimentally determined binding parameters. In agreement with experimental data, the modeling study suggests that whatever was the binding mode of the complex formed, the complexation energy is markedly lower (thus favorable) for AT-containing polynucleotides than for poly d[(G-C)]2. With both poly [d(A).d(T)] and poly [d(A-T)]2, the most energetically favored complex has netropsin and OPC moieties bound simultaneously in the minor groove of DNA. The second favored complex exhibits the bimodal binding, i.e., intercalation of OPC and minor groove binding of the netropsin moiety. For both types of complex, the energy of complex formation is slightly lower with poly [d(A).d(T)]. The binding site sizes of the modeled complexes are about seven and four base pairs to the full groove and bimodal binding, respectively.

Poly(pyrrolecarboxamides) linked to photoactivable chromophore isoalloxazine. Synthesis, selective binding, and DNA cleaving properties.

In an attempt to obtain DNA sequence-specific cleaving molecules, we have synthesized two types of hybrid groove binders composed of an isoalloxazine (flavin) chromophore linked through a polymethylenic chain to either a bis- or a tris(pyrrolecarboxamide) moiety related to netropsin and distamycin, respectively. In both types of molecules, the polymethylenic chain is linked to the alloxazine ring either in the N10 position or in the N3 position. As netropsin and distamycin, the hybrid derivatives preferentially bind to A + T-rich sequences and recognize sequences such as 5'-ATTT. Upon visible light irradiation the flavin moiety undergoes a redox cycling process generating superoxide anion and hydroxyl radical. Generation of oxy radicals appears to be more efficient with the hybrids in which the polymethylenic chain is linked at the N10 position. The generation of oxy radicals results in the occurrence of single strand break in supercoiled DNA. Breaks preferentially occur in the vicinity of A + T-rich sequences. The advantage of flavin relative to other oxy radicals generating compounds such as ferrous-EDTA is that it does not require chemical reduction but can be reduced either by visible light or by cellular enzymes, both conditions being compatible with pharmacological constraints.

Inhibitory effect of the polyanionic drug suramin on the in vitro HIV DNA integration reaction.

An obligatory step in retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely the processing of the LTR ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-1 U5 LTR as substrate and supercoiled pSP65 DNA as target, we have investigated the effect of the polyanionic drug suramin on the catalytic activity of the IN protein. It was found that at stoichiometric suramin to protein ratios, suramin displays a strong inhibitory effect on both the processing and strand transfer reactions. This inhibitory effect is related to the decrease of IN protein binding efficiency to the LTR end DNA fragment.

Effect of topoisomerase inhibitors on the in vitro HIV DNA integration reaction.

Retroviral growth requires as an obligatory step the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the LTR ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-I U5 LTR as substrate and supercoiled pSP65 DNA as target, we have measured the effect of various topoisomerase inhibitors on the functional activity of the IN protein. Among the various drugs tested, the antitumor drug 2N-Methyl, 9-hydroxyellipticinium (NMHE) displays a marked inhibitory effect on the IN-catalyzed U5 insertion. This effect is related to the DNA binding properties of the drug rather than to a selective effect on the IN protein or the DNA-IN protein complex.

Quantitative in vitro assay for human immunodeficiency virus deoxyribonucleic acid integration.

An obligatory step of retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the long terminal repeat (LTR) ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of HIV-1 LTRs as substrate and supercoiled pSP65 DNA as the target, we describe an assay that is suitable for the enzymatic analysis of the integration and for testing candidate inhibitors of HIV IN protein.