Biomedical subjects

G Kukolj

Publications and source records attributed to G Kukolj.

14 recordsLinked to original sources

PubMed2001

Role of DNA end distortion in catalysis by avian sarcoma virus integrase.

Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction). In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction). Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN. Here we used potassium permanganate modification to show that the avian sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro. This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA. Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion. End-distortion activity was also observed with mutant or heterologous DNA substrates. However, further analyses showed that using Mn(2+) as a cofactor, processing site specificity of these substrates was also maintained. Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction. Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.

Avian Sarcoma Viruses↗

PubMed2000

NMR line-broadening and transferred NOESY as a medicinal chemistry tool for studying inhibitors of the hepatitis C virus NS3 protease domain.

This work describes the use of NMR as a medicinal chemistry tool for better understanding the binding characteristics of inhibitors of the HCV NS3 protease. The protease-bound structure of a tetrapeptide-like inhibitor that has an acid C-terminus, a norvaline at P1 and a naphthylmethoxy proline at P2 is described. Conformational comparisons are made with a similar compound having a 1-amino-cyclopropylcarboxylic acid at P1 and with a hexapeptide inhibitor. Differences between the free and bound states are identified. 19F NMR also helped in determining that a single complex is observed when an inhibitor is added to the protease at a 1:1 ratio.

Hepacivirus↗

PubMed2000

The bacteriophage D108 Ner repressor binds a conformationally distinct operator.

The Ner protein encoded by the transposable coliphage D108, an 8.6 kDa lambda Cro-like repressor, binds to an operator spanning 50 bp of DNA. The distinguishing features of this operator are two perfect 11-bp inverted repeats (5'-CCGTGAGCTAC-3') that are separated by an 8-bp AT-rich spacer. Hyperreactivity of the ner operator to potassium permanganate and the hydroxyl radical indicate that the AT-rich spacer assumes a variant conformation consistent with a bend. Using an electrophoretic mobility shift assay, we demonstrated that Ner does not display significant affinity for a single 11-bp site. Furthermore, DNase I protection analysis and circular-permutation binding assays reveal that alterations in the length and sequence of the AT-rich spacer that separates the 11-bp inverted repeats significantly alter Ner-operator interactions, and demonstrate that the intrinsically bent ner operator is conformationally altered upon protein binding.

Bacteriophages↗

PubMed2000

Expression of hTERT and hTR in cis reconstitutes and active human telomerase ribonucleoprotein.

Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3' end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems.

Animals↗

PubMed1999

Solution structure of substrate-based ligands when bound to hepatitis C virus NS3 protease domain.

The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors.

Adenosine Triphosphatases↗

PubMed1999

Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection.

Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.

Animals↗

PubMed1998

Characterization of the nuclear localization signal in the avian sarcoma virus integrase.

A sequence of 21 amino acids (aa) in the C-terminal region of the 286-aa avian sarcoma virus (ASV) integrase (IN) protein has been shown previously to mediate nuclear localization of both IN and beta-galactosidase (betaGal) protein fused to it. This karyophilic sequence includes a high proportion of prolines and residues with basic side chains. In this report, site-directed mutagenesis was used to introduce single aa substitutions of several of these residues. Indirect immunofluorescence showed that IN-betaGal fusion constructs with Ala substitutions for sequence constituents K206, P215, K225 or R227 had lost the exclusive nuclear localization capability of the wild-type fusion. A fusion protein with the conservative substitution K206R retained the nuclear localization capacity. The site-specific substitutions that reduced karyophilic activity had no effect on the processing or joining activities of IN in vitro. However, the introduction of three of the four Ala codon substitutions into viral DNA clones caused a significant delay in viral replication following transfection of cycling chicken embryo fibroblasts. These results are consistent with a possible role for ASV IN in nuclear targeting.

Amino Acid Sequence↗

PubMed1997

Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases.

The composition and subcellular trafficking of subviral preintegration complexes are reported to vary among the different retroviruses. The process by which the avian sarcoma virus (ASV) preintegration complex gains access to target chromatin remains unknown. Here we report that ASV integrase (IN) expressed as a fusion to beta-galactosidase accumulates in the nuclei of transfected COS-1 cells. In contrast, human immunodeficiency type 1 (HIV-1) IN-beta-galactosidase fusions expressed similarly are predominantly cytoplasmic. To identify the region of ASV IN that specifies nuclear localization, various subdomains of the protein were expressed as beta-galactosidase fusions and their subcellular locations were assessed cytochemically and by indirect immunofluorescence. These analyses showed that the ASV IN protein possesses a functional nuclear localization signal that spans amino acids 206 to 235 and displays limited homology with known nuclear transport signals.

Amino Acid Sequence↗

PubMed1995

Enhanced and coordinated processing of synapsed viral DNA ends by retroviral integrases in vitro.

We have designed novel substrates to investigate the first step in retroviral integration: the site-specific processing of two nucleotides from the 3' ends of viral DNA. The substrates consist of short duplex oligodeoxynucleotides whose sequences match those of the U3 and U5 ends of viral DNA but are covalently synapsed across the termini by short, single-strand nucleotide linkers. We show here that the optimal separation between termini in a synapsed-end substrate for avian sarcoma/leukosis virus (ASV) IN is 2 nucleotides. This places the two conserved 5'-CA-3' processing sites 6 nucleotides apart, a separation equal to the staggered cut in target DNA produced by this enzyme during the subsequent joining reaction. Based on estimates of initial reaction rates, this synapsed-end substrate is processed by IN at > 10-fold higher efficiency than observed with an equivalent mixture of U3 and U5 single-end (uncoupled) substrates. Enhanced processing is maintained at low IN concentrations, suggesting that the synapsed-end substrate may facilitate enzyme multimerization. Enhanced processing by HIV-1 IN, which produces a 5-bp stagger during integration, was observed with a synapsed-end substrate in which the separation between processing sites was 5 nucleotides. These observations provide estimates of the distances between active sites in the multimeric IN-DNA complexes of ASV and HIV-1. Our results also show that processing of paired U3 and U5 ends need not be coupled temporally. Finally, we observed that substrates that paired a wild-type with a mutated terminus were cleaved poorly at both ends. Thus, in vitro processing of the synapsed-end substrates requires specific recognition of the sequences at both ends. These findings provide new insights into the mechanism of integrative recombination by retroviral integrases and, by extension, other prokaryotic and eukaryotic transposases that are related to the viral enzymes.

Avian Sarcoma Viruses↗

PubMed1994

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: evidence for altered protein and DNA structures in the complex.

Ner repressors of the transposable phages Mu and D108 play a central role in regulating the expression of the early (transposase) operon and in ensuring that phage growth proceeds along a lytic pathway. The latter function is analogous to that performed by the Cro protein of phage lambda. Unlike lambda Cro, however, the structural basis of operator recognition is not known for the Ner repressors. In order to elucidate the structural features underlying operator recognition by Ner repressors, we have employed Raman spectroscopy as a probe of the solution secondary structures of both D108 Ner and Mu Ner. Additionally, we have obtained Raman spectra of the D108 Ner repressor when bound to a 61-base-pair oligodeoxynucleotide containing the 55-base-pair D108 ner binding site. Conformation-sensitive Raman bands show that both D108 and Mu Ner contain similar, highly alpha-helical (approximately 45%) secondary structures. The Raman markers also show that the substantial nonhelical secondary structure of both D108 Ner and Mu Ner is largely beta-stranded. The protein-free 61-bp D108 ner operator exhibits Raman marker bands diagnostic of an uninterrupted B DNA duplex. In the D108 Ner:DNA complex, we find the following: (i) B DNA stereochemistry is fully conserved, although with significant perturbations to the B form backbone geometry, particularly in AT-rich regions of the bound operator. (ii) The specific interactions that occur between Ner repressor and operator involve B DNA major groove sites. (iii) A small (8 +/- 3%) increase in alpha-helix content of the Ner repressor is detected upon operator binding. (iv) Finally, the local environments of many aromatic amino acids are substantially altered in the D108 Ner:DNA complex. We propose a molecular model for binding of D108 Ner to its operator that is consistent with both the present spectroscopic findings and the results of recent biochemical studies. Essential features of this model are bending of the DNA double helix and contact of operator sites with repressor domains bearing sequence homologies with the helix-turn-helix (HTH) motifs of other DNA-binding proteins. The Raman fingerprint of the Ner:DNA complex is shown to be clearly distinguishable from that of the lambda cI:DNA complex, even though both gene regulatory complexes are presumed to employ HTH recognition motifs. The unique Raman signatures observed for these repressor complexes suggest that the Raman methodology may be useful in discriminating different modes of operator recognition by the HTH motifs of regulatory proteins.

Amides↗

PubMed1992

Integration host factor activates the Ner-repressed early promoter of transposable Mu-like phage D108.

The lytic-lysogenic switch in transposable, Mu-like bacteriophage D108 is governed by two divergent and slightly overlapping transcription units originating from the Pe and Pc promoters. DNase I footprinting and in vivo mutational analysis suggest that lysogeny is maintained by c-repressor occupancy of the O2 operator, which precludes RNA polymerase from binding to Pe. Lytic development is controlled by the Ner repressor, which binds to a site symmetrically situated between the converging promoters and, in the absence of other factors, prevents RNA polymerase from binding to either Pc or Pe. DNase I protection and potassium permanganate hypersensitivity in the presence of integration host factor (IHF), which binds and alters the DNA structure upstream of Pe, revealed that RNA polymerase was able to bind Pe irrespective of the Ner.DNA-bound complex, and partially unwind the Pe "-10 region." Ner repression of Pe transcription in vitro was significantly more effective in the absence of IHF. Using a cloned D108 early region-lacZ fusion in IHF-deficient and -proficient backgrounds, we also demonstrate this host factor's affect on ner-repressed Pe in vivo, and generate a system for isolating mutants in the regulatory genes and sites controlling this genetic switch. D108 lytic growth is proposed to occur through IHF-mediated activation of the phage Ner-repressed early operon.

Amino Acid Sequence↗

PubMed1991

Characterization of the lysogenic repressor (c) from transposable Mu-like bacteriophage D108.

The c gene products from related, transposable phages Mu and D108 encode lysogenic repressors which negatively regulate transcription and transposition. Using the gel shift assay to monitor c-operator specific DNA-binding activity, the 19.5 kDa D108 c repressor was purified to homogeneity. Sequence analysis of the N-terminus confirmed the identity of the purified protein as the repressor and ascribed its ATG initiation codon to base pair 864 from the D108 left end. Analytical gel filtration and dimethyl suberimidate cross-linking of repressor at 0.1-0.5 microM concentrations revealed that the repressor protein could form oligomers in the absence of its DNA substrate. From DNase I footprinting and gel mobility shift analyses, the D108 repressor only bound to two operators (O1 and O2) which, as in Mu, flank an Integration Host Factor (IHF) binding site. In contrast to Mu, an O3 site in D108 was not found. Moreover, D108 repressor first bound operator O2, while occupancy of O1 required higher protein concentrations. The implications of these results on the D108 regulatory system are discussed.

Bacteriophage mu↗

PubMed1989

Purification and characterization of the Ner repressor of bacteriophage Mu.

The Ner protein of bacteriophage Mu acts as a lambda cro-like negative regulator of the phage's early (transposase) operon. Using the band retardation assay to monitor ner-operator-specific DNA-binding activity, the 8 kDa Ner protein was purified to homogeneity. DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5'-ANPyTAPuCTAAGT-3', separated by a 6 bp spacer region. Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner. Unlike the functionally similar lambda cro protein, gel filtration experiments show that the native molecular mass of Mu Ner to be approx. 8 kDa. These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.

Base Sequence↗

PubMed1989

DNA-directed oligomerization of the monomeric Ner repressor from the Mu-like bacteriophage D108.

We have purified the 8.6 kd ner gene product (a lambda Cro-like protein which negatively regulates transcription from two divergent and overlapping promoters) from the Mu-like transposable bacteriophage D108. Chemical and enzymatic protection experiments show the D108 ner-operator to contain two perfect 11 bp (5'-CCG-TGAGCTAC-3') inverted repeats separated by an 8 bp AT-rich region. Ner makes base-specific contacts in the major groove spanning the 11 bp repeats and also interacts with regions flanking these sites such that its operator comprises five turns of the DNA helix. Furthermore, gel filtration chromatography and dimethyl suberimidate crosslinking experiments indicate that D108 Ner (at concentrations exceeding 5 microM) is a monomer in solution, yet crosslinks as a dimer when bound to its operator site. As a small (73 amino acids) monomeric protein, Ner does not display strong homology with any known DNA-binding proteins. By virtue of the interactions with its operator it appears to bind DNA in a markedly different manner from other known prokaryotic repressors thus adding to the growing catalog of protein motifs used for specific binding to DNA.

Bacteriophage mu↗