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A Mazumder

Publications and source records attributed to A Mazumder.

At least 37 records · Page 2Linked to original sources
PubMed1998

The B-subunit of cholera toxin induces immunoregulatory cells and prevents diabetes in the NOD mouse.

The B-subunit of the cholera toxin molecule (CT-B) has T-cell immunomodulatory properties. Because the pathogenesis of diabetes in the nonobese diabetic (NOD) mouse model of IDDM is thought to be a T-cell-mediated process due to an imbalance of immunoregulatory and anti-islet effector cells, we examined the effect of CT-B administration on the development of diabetes in the NOD mouse and assessed whether this potential diabetes-sparing effect of CT-B is mediated by changes in immunoregulatory and/or anti-islet cytotoxic effector cell activity. The administration of either intravenous or intraperitoneal CT-B decreased the development of diabetes with no apparent drug toxicity. At 6 months of age, only 18% of CT-B vs. 75% of saline-treated animals had diabetes. Histopathological examination revealed less islet atrophy in CT-B-treated animals. The in vitro proliferative responses of mononuclear splenocytes and thymocytes to concanavalin A and lipolysaccharide and the proportion of B-cells and T-cell subsets were not altered by CT-B treatment. CT-B administration did not inhibit the primary immunization of mice to tetanus toxoid. The development of diabetes in irradiated NOD mice was slower in the animals injected with spleen cells (SC) from CT-B-treated than from saline-treated NOD mice, suggesting that CT-B decreases anti-islet effector cell activity. The injection of SC from CT-B-treated mice inhibited the adoptive transfer of diabetes by SC from diabetic mice into irradiated NOD mice, documenting that CT-B administration induces regulatory cell activity. In conclusion, CT-B administration prevents the development of diabetes in NOD mice by inhibiting the immune destruction of islets. This islet-sparing activity appears mediated, at least in part, by the induction of regulatory cells and, in turn, suppression of anti-islet effector cells, which is not associated with generalized immunosuppression or T- or B-cell depletion.

Adoptive Transfer↗
PubMed1997

Trapping of mammalian topoisomerase I and recombinations induced by damaged DNA containing nicks or gaps. Importance of DNA end phosphorylation and camptothecin effects.

We used purified mammalian topoisomerases I (top1) and oligonucleotides containing a unique top1 cleavage site to study top1-mediated cleavage and recombination in the presence of nicks and short gaps mimicking DNA damage. In general, top1 cleavage was not induced opposite to the nicks, and nicks upstream from the top1 cleavage site suppressed top1 activity. Irreversible top1 cleavage complexes ("suicide products" or "aborted complexes") were produced in DNA containing nicks or short gaps just opposite to the normal top1 cleavage site. Camptothecin enhanced the formation of the aborted top1 complexes only for nicks downstream from the cleavage site. These aborted (suicide) complexes can mediate DNA recombination and promote illegitimate recombination by catalyzing the ligation of nonhomologous DNA fragments (acceptors). We report for the first time that top1-mediated recombination is greatly enhanced by the presence of a phosphate at the 5' terminus of the top1 aborted complex (donor DNA). By contrast, phosphorylation of the 3' terminus of the gap did not affect recombination. At concentrations that strongly enhanced inhibition of intramolecular religation, resulting in an increase of top1 cleavable complexes, camptothecin did not reduce recombination (intermolecular religation). Nicks or gaps with 5'-phosphate termini would be expected to be produced directly by ionizing radiations or by processing of abasic sites and DNA lesions induced by carcinogens or drugs used in cancer chemotherapy. Thus, these results further demonstrate that DNA damage can efficiently trap top1-cleavable complexes and enhance top1-mediated DNA recombination.

Camptothecin↗
PubMed1997

Curcumin analogs with altered potencies against HIV-1 integrase as probes for biochemical mechanisms of drug action.

We have previously reported the inhibitory activity of curcumin against human immunodeficiency virus type one (HIV-1) integrase. In the present study, we have synthesized and tested analogs of curcumin to explore the structure-activity relationships and mechanism of action of this family of compounds in more detail. We found that two curcumin analogs, dicaffeoylmethane (6) and rosmarinic acid (9), inhibited both activities of integrase with IC50 values below 10 microM. We have previously demonstrated that lysine 136 may play a role in viral DNA binding. We demonstrated equivalent potencies of two curcumin analogs against both this integrase mutant and wild-type integrase, suggesting that the curcumin-binding site and the substrate-binding site may not overlap. Combining one curcumin analog with the recently described integrase inhibitor NSC 158393 resulted in integrase inhibition which was synergistic, reflective of drug-binding sites which may not overlap. We have also determined that these analogs can inhibit binding of the enzyme to the viral DNA but that this inhibition is independent of divalent metal ion. Furthermore, kinetic studies of these analogs suggest that they bind to the enzyme at a slow rate. These studies can provide mechanistic and structural information which may guide the future design of integrase inhibitors.

4-Hydroxycoumarins↗
PubMed1997

Arylamide inhibitors of HIV-1 integrase.

Based on data derived from a large number of HIV-1 integrase inhibitors, similar structural features can be observed, which consist of two aryl units separated by a central linker. For many inhibitors fitting this pattern, at least one aryl ring also requires ortho bis-hydroxylation for significant inhibitory potency. The ability of such catechol species to undergo in situ oxidation to reactive quinones presents one potential limitation to their utility. In an effort to address this problem, a series of inhibitors were prepared which did not contain ortho bishydroxyls. None of these analogues exhibited significant inhibition. Therefore an alternate approach was taken, whose aim was to increase potency rather than eliminate catechol substructures. In this latter study, naphthyl nuclei were utilized as aryl components, since a previous report had indicated that fused bicyclic rings may afford higher affinity relative to monocyclic phenyl-based systems. In preliminary work with monomeric units, it was found that the 6,7-dihydroxy-2-naphthoic acid (17) (IC50 = 4.7 microM) was approximately 10-fold more potent than its 5,6-dihydroxy isomer 19 (IC50 = 62.4 microM). Of particular note was the dramatic difference in potency between free acid 17 and its methyl ester 21 (IC50 > 200 microM). The nearly total loss of activity induced by esterification strongly indicates that the free carboxylic -OH is important for high potency of this compound. This contrasts with the isomeric 5,6-dihydroxy species 19, where esterification had no effect on inhibitory potency (23, IC50 = 52.7 microM). These data provide evidence that the monomeric 6,7- and 5,6-dihydroxynaphthalenes may be interacting with the enzyme in markedly different fashions. However, when these naphthyl nuclei were incorporated into dimeric structures, significant enhancements in potencies each relative to the monomeric acids were observed, with bis-6,7-dihydroxy analogue 49 and bis-5,6-dihydroxy analogue 51 both exhibiting approximately equal potencies (IC50 values of 0.81 and 0.11 microM, respectively).

Caffeic Acids↗
PubMed1997

Effects of uracil incorporation, DNA mismatches, and abasic sites on cleavage and religation activities of mammalian topoisomerase I.

Abasic sites and deamination of cytosine to uracil are probably the most common types of endogenous DNA damage. The effects of such lesions on DNA topoisomerase I (top1) activity were examined in oligonucleotides containing a unique top1 cleavage site. The presence of uracils and abasic sites within the first 4 bases immediately 5' to the cleavage site suppressed normal top1 cleavage and induced new top1 cleavage sites. Uracils immediately 3' to the cleavage site increased cleavage and produced a camptothecin mimicking effect. A mismatch with a bulge or abasic sites immediately 3' to the top1 cleavage site irreversibly trapped top1 cleavable complexes in the absence of camptothecin and produced a suicide cleavage complex. These results demonstrate that top1 activity is sensitive to physiological, environmental, and pharmacological DNA modifications and that top1 can act as a specific mismatch- and abasic site-nicking enzyme.

Animals↗
PubMed1997

HIV-1 integrase pharmacophore: discovery of inhibitors through three-dimensional database searching.

Starting from a known inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN); caffeic acid phenethyl ester (CAPE), a putative three-point pharmacophore for binding of inhibitors to IN was derived. This pharmacophore was used to search the National Cancer Institute three-dimensional (3D) structural database. Out of the open, nonproprietary part of this database, comprising approximately 200000 compounds, 267 structures were found to match the pharmacophore in at least one conformation, and 60 of those were tested in an in vitro assay against HIV-1 IN. Out of these, 19 were found to inhibit both the 3'-processing and strand transfer of IN at micromolar concentrations. In order to test the validity of this pharmacophore, a small 3D database of 152 published IN inhibitors was built. A search in this database yielded a statistically significant correlation of the presence of this pharmacophore and the potency of the compounds. An automated pharmacophore identification procedure performed on this set of compounds provided additional support for the importance of this pharmacophore for binding of inhibitors to IN and hinted at a possible second pharmacophore. The role of aromatic moieties in the binding of ligands to HIV-1 IN through interactions with divalent metal cations, which are known to be necessary for activity of the enzyme, was explored in ab initio calculations.

Caffeic Acids↗
PubMed1997

Discovery of HIV-1 integrase inhibitors by pharmacophore searching.

Based upon a class of known HIV-1 integrase inhibitors, several pharmacophore models were proposed from molecular modeling studies and validated using a 3D database of 152, compounds for which integrase assay data are known. Using the most probable pharmacophore model as the query, the NCI 3D database of 206,876 compounds was searched, and 340 compounds that contain the pharmacophore query were identified. Twenty-nine of these compounds were selected and tested in the HIV-1 integrase assay. This led to the discovery of 10 novel, structurally diverse HIV-1 integrase inhibitors, four of which have an IC50 value less than 30 microM and are promising lead compounds for further HIV-1 integrase inhibitor development.

4-Hydroxycoumarins↗
PubMed1997

Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching.

Seventeen lichen acids comprising despides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200,000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 microM; 15 were below 50 microM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.

Anti-HIV Agents↗
PubMed1997

Coumarin-based inhibitors of HIV integrase.

The structures of a large number of HIV-1 integrase inhibitors have in common two aryl units separated by a central linker. Frequently at least one of these aryl moieties must contain 1,2-dihydroxy substituents in order to exhibit high inhibitory potency. The ability of o-dihydroxy-containing species to undergo in situ oxidation to reactive quinones presents a potential limitation to the utility of such compounds. The recent report of tetrameric 4-hydroxycoumarin-derived inhibitor 5 provided a lead example of an inhibitor which does not contain the catechol moiety. Compound 5 represents a large, highly complex yet symmetrical molecule. It was the purpose of the present study to determine the critical components of 5 and if possible to simplify its structure while maintaining potency. In the present study, dissection of tetrameric 5 (IC50 = 1.5 microM) into its constituent parts showed that the minimum active pharmacophore consisted of a coumarin dimer containing an aryl substituent on the central linker methylene. However, in the simplest case in which the central linker aryl unit consisted of a phenyl ring (compound 8, IC50 = 43 microM), a significant reduction in potency resulted by removing two of the original four coumarin units. Replacement of this central phenyl ring by more extended aromatic systems having higher lipophilicity improved potency, as did the addition of 7-hydroxy substituents to the coumarin rings. Combining these latter two modifications resulted in compounds such as 3,3'-(2-naphthalenomethylene)bis[4,7-dihydroxycoumarin] (34, IC50 = 4.2 microM) which exhibited nearly the full potency of the parent tetramer 5 yet were structurally much simpler.

Anti-HIV Agents↗
PubMed1997

Effect of the in vivo priming regimen for peripheral blood stem cells (PBSC) mobilization on in vitro generation of cytotoxic effectors by IL-2 activation of PBSC in a murine model.

Priming of patients with different PBSC mobilizing regimens leads to an increase by several fold in circulating hematopoietic progenitors in peripheral blood. However, the effect of these mobilizing regimens on lymphoid cells contained within the harvested PBSC population is not well understood. We have studied the effect of CY and/or G-CSF +/- IL-2 containing regimens on lymphoid cells, and their capacity to give rise to cytotoxic effectors on subsequent in vitro IL-2 activation in a murine model of PBSC mobilization. C57B1/6 mice were given CY 100 or 200 mg/kg on day 0 followed 48 h later by G-CSF 125 micrograms/kg twice a day and/or IL-2 60000 i.u. twice a day in different schedules. Mice were sacrificed on day 4, 6, 8 and 10 following CY and the number of hematopoietic progenitors mobilized to the spleens of these mice was assessed by CFC assay and cytotoxicity was evaluated by 4 h 51Cr release assay against both NK-sensitive (Yak-1), and NK-resistant (B16, C1498) cell lines after 24 h in vitro IL-2 activation in the presence of 6000 i.u./ml of IL-2. Peak numbers of CFC in the splenic PBSC population were seen on day 6 following CY. Administration of CY 200 mg/kg + GCSF, the most potent regimen for CFC mobilization, led to a marked decrease in proportion of CD3+ cells in day 6 PBSC as compared to controls (17.7% vs 3.9%) and was associated with a significant decrease in generation of cytotoxic cells after IL-2 activation. Combining IL-2 to CY + G-CSF prevented the marked loss in cytotoxicity associated with this regimen without any decrease in number of CFC mobilized. When IL-2 was combined with CY without G-CSF, the number of CFC mobilized was comparable to that seen with CY + G-CSF and these CY + IL-2 mobilized PBSC generated potent cytotoxic effectors after in vitro IL-2 activation. Thus our results indicate that combining IL-2 with a PBSC mobilizing regimen can avert a decrease in the cytotoxic potential of mobilized cells without compromising the number of hematopoietic progenitors.

Animals↗
PubMed1997

Suppression of progenitor cell growth by vancomycin following autologous stem cell transplantation.

The occurrence of hematologic side-effects resulting from the use of vancomycin is rare. Prior to this report, vancomycin-induced neutropenia was believed to be due to a hypersensitivity reaction since antibodies directed against circulating neutrophils have been discovered in the serum of some patients. We demonstrate suppression of hematopoietic bone marrow progenitor cells in a patient experiencing vancomycin-induced neutropenia after an autologous hematopoietic stem cell transplantation for multiple myeloma. A bone marrow (BM) specimen obtained at the time of neutropenia demonstrated direct suppression of progenitor cell growth in vitro when vancomycin was added at increasing concentrations (1, 10 and 50 microg/ml). No such trend was noted in a BM sample from the same patient obtained 11 months prior to transplantation and a normal control BM. The decrease in the total number of colony-forming units (CFU) was statistically significant at all the dose levels of vancomycin when compared to the number of CFU in the baseline BM sample (P < 0.05). The myeloid maturation arrest observed in the bone marrow sample obtained during the period of neutropenia and the dose dependent growth inhibition by vancomycin observed in vitro suggest a novel nonimmune mechanism of hematologic effects due to suppression of bone marrow progenitor cell growth.

Anti-Bacterial Agents↗
PubMed1997

Interleukin-2-activated hematopoietic stem cell transplantation for breast cancer: investigation of dose level with clinical correlates.

Incubating hematopoietic stem cells with IL-2 in vitro for 24 h generates cytotoxic T cells. When infused into patients, these cells may stimulate a graft-versus-tumor (GVT) effect. This clinical trial was designed to assess the ability of IL-2 activated peripheral blood stem cells (PBSC) to reconstitute hematopoiesis, to investigate dose levels and dose-limiting toxicities of IL-2, and to evaluate clinical results and preliminary laboratory effects using a combination of IL-2-activated autologous PBSC followed by IL-2 after transplantation. Sixty-one women with stage II-IV breast cancer were treated. After the administration of carboplatin (200 mg/m2/day for 3 days) and cyclophosphamide (2 g/m2/day for 3 days), patients received autologous PBSC that were cultured in IL-2 for 24 h followed by parenteral administration of IL-2 beginning the day of transplantation. Three escalating doses of IL-2 were evaluated with increasing duration up to 4 weeks. Of the 57 patients receiving IL-2 after tranplantation, 19 patients (33.3%) were unable to complete the planned course of IL-2 therapy due to persistent fevers (n = 9), diarrhea (n = 2), pulmonary capillary leak syndrome (n = 3), development of a rash (n = 1), atrial fibrillation (n = 1), or patient's request (n = 3). One death occurred during hospitalization. Engraftment of neutrophils occurred on day 11.5 (mean; range 8-21 days) and platelets on day 11.7 (mean; range 7-33 days). The maximal tolerated dose of IL-2 was 6 x 10(5) IU/m2/day for 4 weeks. Disease-free survival rates for all stages were comparable to current reports in the literature. Preliminary laboratory evaluations include FACScan analysis of the IL-2 activated PBSC demonstrating an increased percentage of CD3+, CD25+, HLA-DR+ T cells. Phenotypically similar cells were present in peripheral blood samples of patients when tested 15 days after transplantation. This study demonstrates successful engraftment with IL-2-activated PBSC after high-dose chemotherapy for women with stage II-IV breast cancer. The regimen is feasible and, although toxicities are common, they are manageable and correlate with increasing dose and duration of IL-2.

Adult↗
PubMed1997

A pilot study evaluating interleukin-2-activated hematopoietic stem cell transplantation for hematologic malignancies.

Cytotoxic effector cells are generated when autologous hematopoietic cells (HSC) are cultured with IL-2 for 24 h. Infusion of these cells followed by IL-2 administration may moderate a graft-versus tumor (GvT) effect in vivo. Sixteen patients--7 with non-Hodgkin's lymphoma (NHL), 2 with AML, 4 with multiple myeloma (MM), and 3 with Hodgkin's disease (HD)--received busulfan (4 mg/kg/day for 4 days) and cyclophosphamide (60 mg/kg/day for 2 days) or cyclophosphamide/TBI (1320 cGy). Autologous HSC were activated by culturing with IL-2 for 24 h before reinfusion. Subcutaneous administration of IL-2 began after engraftment at 1.8 x 10(6) IU/m2/day for 1-4 weeks. Neutrophil engraftment occurred on day 13.1 (median) (range 9-45 days), and platelet engraftment occurred on day 19.3 (median) (range 7-54 days) for 15 patients, with delayed engraftment observed in 3 patients. One patient experienced a fatal cardiac arrhythmia. Five patients developed transient skin rashes with histologic evaluation demonstrating findings consistent with GvHD. At 17 months (median) (range 7-23 months), 9 patients are alive, with 6 patients remaining disease free. This pilot trial demonstrates mild to moderate toxicities and a possible delay in platelet engraftment. Further trials will determine the optimal dose and duration of IL-2 therapy and possible impact of this therapy on survival. Laboratory investigations are evaluating the presence of autologous GvHD and a possible GvT effect.

Adolescent↗
PubMed1997

Probing interactions between viral DNA and human immunodeficiency virus type 1 integrase using dinucleotides.

Retroviral integrases are essential for viral replication and represent an attractive chemotherapeutic target. In the current study, we demonstrated the activity of micromolar concentrations of dinucleotides against human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), simian immunodeficiency virus, and feline immunodeficiency virus integrases. The structure-activity relationship indicates that 5'-phosphorylation enhances potency and that phosphodiester and sugar modifications affect the inhibition of HIV-1 integrase. Base sequence selectivity was observed: pAC, pAT, and pCT were the most potent inhibitors, whereas pAA, pGA, and pGC showed low activity at 100 microM. The inhibition by pAC is consistent with the interaction of the enzyme with the 5' end of the noncleaved strand (5'-AC-3'). The linear and cyclic dinucleotides released by the 3'-processing reaction did not affect enzymatic activity at physiological concentrations. An increase in the length to trinucleotides or tetranucleotides enhanced potency by only 2-3-fold, suggesting that two neighboring bases may be sufficient for significant interactions. Inhibition of a truncated (50-212) integrase mutant and global inhibition of all nucleophiles in the 3'-processing reaction suggest that dinucleotides bind in the catalytic core. All of the active dinucleotides inhibited enzyme/DNA binding in their respective IC50 range. Although the dinucleotides tested showed no antiviral activity, these observations demonstrate the usefulness of dinucleotides in elucidating enzyme mechanisms and as potential ligands for cocrystallization and as lead structures for development of antivirals.

Animals↗
PubMed1997

Diarylsulfones, a novel class of human immunodeficiency virus type 1 integrase inhibitors.

A majority of reported human immunodeficiency virus type 1 integrase (HIV-1 IN) inhibitors are polyhydroxylated aromatic compounds containing two phenyl rings separated by aliphatic or aromatic linkers. Most inhibitors possessing a catechol moiety exhibit considerable toxicity in cellular assays. In an effort to identify nonhydroxylated analogs, a series of aromatic sulfones were tested for their ability to inhibit the 3' processing and strand transfer steps that are necessary for HIV replication. Several aromatic sulfones have previously been shown to have moderate activity against HIV-1 reverse transcriptase in cellular assays; however, their inhibitory potencies against IN have not been explored. In the present study, the inhibitory effect of a series of sulfones and sulfonamides against IN was determined. Among 52 diaryl sulfones tested, 4 were determined to be highly potent (50% inhibitory concentration [IC50], 0.8 to 10 micrograms/ml), 5 had good potencies (IC50, 11 to 50 micrograms/ml), 10 showed moderate potencies (IC50, 51 to 100 micrograms/ml), and 33 were inactive (IC50, > 100 micrograms/ml) against IN. All of the active compounds exhibited similar potencies against HIV-2 IN. Sulfa drugs, used extensively in treating Pneumocystis carinii pneumonia, a leading cause of morbidity and mortality in AIDs patients, were also examined. Among 19 sulfonamides tested, sulfasalazine (IC50, 50 micrograms/ml) was the most potent. We conclude that potent inhibitors of IN can be designed based on the results presented in this study.

Cell Line↗
PubMed1997

Experimental evidence of interleukin-2 activity in bone marrow transplantation.

PURPOSE: This article reviews the preclinical and clinical experience with the administration of recombinant interleukin-2 (rIL-2) and rIL-2-activated lymphocytes following allogeneic or autologous bone marrow transplantation in patients with hematologic malignancies and advanced breast cancer. METHODS: Recombinant IL-2 was administered following engraftment of bone marrow or peripheral blood stem cell. (PBSC) transplants to enhance the graft versus tumor effect. Ex vivo incubation of bone marrow or PBSC with rIL-2 was used to activate effector lymphocytes (i.e., T cells and natural killer cells) that may enhance the antitumor effect. RESULTS: We have shown in preclinical models that the incubation of bone marrow or PBSC in rIL-2 generates cytotoxic effector cells that are capable of lysing a wide variety of tumor cells. In those models we have also found that these same rIL-2-activated cells are able to induce a syngeneic graft-versus-host disease (GVHD) that correlates with the antitumor effect. We have now taken this concept into the clinic in patients with hematologic malignancies and advanced breast cancer. We have treated patients with stage II through stage IV breast cancer with PBSC that have been incubated with rIL-2. We have now reached a stable phase II dose of rIL-2 with no adverse effect on engraftment. We have found that cytotoxic cells are generated in the patients' circulation, and that these cells are activated T cells, not natural killer cells. Approximately 50% of the patients seem to have autologous GVHD. Patients with stage II breast cancer seem to generate more cytotoxic cells than patients with stage IV breast cancer. We are in the process of examining whether these patients have an increased disease-free survival. CONCLUSION: We have shown that rIL-2-activated cells seem to generate autologous GVHD and possibly induce a graft-versus-tumor effect. Use of similar approaches in BMT will be important in the future to generate similar types of immune antitumor effects.

Breast Neoplasms↗
PubMed1996

Chemical trapping of ternary complexes of human immunodeficiency virus type 1 integrase, divalent metal, and DNA substrates containing an abasic site. Implications for the role of lysine 136 in DNA binding.

We report a novel assay for monitoring the DNA binding of human immunodeficiency virus type 1 (HIV-1) integrase and the effect of cofactors and inhibitors. The assay uses depurinated oligonucleotides that can form a Schiff base between the aldehydic abasic site and a nearby enzyme lysine epsilon-amino group which can subsequently be trapped by reduction with sodium borohydride. Chemically depurinated duplex substrates representing the U5 end of the HIV-1 DNA were initially used. We next substituted an enzymatically generated abasic site for each of 10 nucleotides normally present in a 21-mer duplex oligonucleotide representing the U5 end of the HIV-1 DNA. Using HIV-1, HIV-2, or simian immunodeficiency virus integrases, the amount of covalent enzyme-DNA complex trapped decreased as the abasic site was moved away from the conserved CA dinucleotide. The enzyme-DNA complexes formed in the presence of manganese were not reversed by subsequent addition of EDTA, indicating that the divalent metal required for integrase catalysis is tightly bound in a ternary enzyme-metal-DNA complex. Both the N- and C-terminal domains of integrase contributed to efficient DNA binding, and mutation of Lys-136 significantly reduced Schiff base formation, implicating this residue in viral DNA binding.

Base Sequence↗