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A Bochkarev

Publications and source records attributed to A Bochkarev.

13 recordsLinked to original sources
PubMed1998

The 2.2 A structure of a permanganate-sensitive DNA site bound by the Epstein-Barr virus origin binding protein, EBNA1.

Epstein-Barr nuclear antigen 1 (EBNA1) binds to four recognition sites in the minimal origin of latent DNA replication of Epstein-Barr virus and activates latent-phase replication of the viral genomes. Two of these EBNA1 binding sites become sensitive to permanganate oxidation when bound by the DNA binding and dimerization domains of EBNA1. We have previously solved the co-crystal structure of this EBNA1 fragment bound to a consensus recognition site that is not sensitive to permanganate oxidation (CS). To understand the structural difference that underlies the permanganate sensitivity of EBNA1 binding sites, we have now solved the crystal structure of the EBNA1 DNA-binding and dimerization domains bound to a permanganate-sensitive site (CSA/T). Comparisons of permanganate-sensitive and insensitive EBNA1-DNA complexes have revealed only minor differences in protein and DNA structures. In the EBNA1-CSA/T structure, interstrand H-bonds for three consecutive base-pairs centered over the permanganate-sensitive thymine base are lengthened relative to the corresponding bonds in the EBNA1-CS complex, and three potential intrastrand H-bonds were observed between adjacent bases. We also observed that both the CS and CSA/T sequences are overwound by EBNA1 in the vicinity of the permanganate-sensitive thymine base. Finally, we show that the permanganate-sensitive thymine base in the CSA/T-EBNA1 complex is more accessible to solvent than the corresponding T in the EBNA-CS complex.

Binding Sites↗
PubMed1998

The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain.

Replication protein A (RPA) is a conserved nuclear single-stranded DNA (ssDNA)-binding protein. Human RPA (hRPA) comprises three subunits of approximately 70, 32, and 14 kDa (hRPA70, hRPA32 and hRPA14). RPA is known to bind ssDNA through two ssDNA-binding domains in the RPA70 subunit. Here, we demonstrate that the complex of hRPA32 and hRPA14 has an ssDNA-binding domain. Limited proteolysis of the hRPA14.32 complex defined a core dimer composed of the central region of hRPA32 (amino acids 43-171) and RPA14. The core dimer bound ssDNA with an affinity of approximately 10-50 microM, which is at least 100-fold more avid than the DNA-binding affinity of the intact dimer. Analysis of the predicted secondary structure of hRPA32 suggests that amino acids 63-150 of hRPA32 form an ssDNA-binding domain similar in structure to each of those in hRPA70. The complex of hRPA14 and hRPA32-(43-171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436-616). The ssDNA-binding affinity of this trimeric complex was 3 to 5-fold higher than hRPA14.32-(43-171) alone, suggesting a role for the C terminus of hRPA70 in ssDNA binding.

Amino Acid Sequence↗
PubMed1998

Origin DNA-binding proteins.

The first step in DNA replication involves the recognition of origin DNA sequences by origin-binding proteins. The three-dimensional structures of three different origin DNA-binding proteins have recently been solved. These proteins form a structural class distinct from other DNA-binding proteins. One of the origin-binding proteins, Epstein-Barr virus nuclear antigen 1, most likely has two modes of DNA binding; the sequential use of these modes may be important for the initiation of DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed1997

Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA.

The single-stranded-DNA-binding proteins (SSBs) are essential for DNA function in prokaryotic and eukaryotic cells, mitochondria, phages and viruses. The structures of four SSBs have been solved, but the molecular details of the interaction of SSBs with DNA remain speculative. We report here the crystal structure at 2.4 A resolution of the single-stranded-DNA-binding domain of human replication protein A (RPA) bound to DNA. Replication protein A is a heterotrimeric SSB that is highly conserved in eukaryotes. The largest subunit, RPA70, binds to single-stranded (ss)DNA and mediates interactions with many cellular and viral proteins. The DNA-binding domain, which lies in the middle of RPA70, comprises two structurally homologous subdomains oriented in tandem. The ssDNA lies in a channel that extends from one subdomain to the other. The structure of each RPA70 subdomain is similar to those of the bacteriophage SSBs, indicating that the mechanism of ssDNA-binding is conserved.

Amino Acid Sequence↗
PubMed1997

Replication protein A. Characterization and crystallization of the DNA binding domain.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein in eukaryotic cells. The DNA binding activity of human RPA has been previously localized to the N-terminal 441 amino acids of the 70-kDa subunit, RPA70. We have used a combination of limited proteolysis and mutational analysis to define the smallest soluble fragment of human RPA70 that retains complete DNA binding activity. This fragment comprises residues 181-422. RPA181-422 bound DNA with the same affinity as the 1-441 fragment and had a DNA binding site of 8 nucleotides or less. RPA70 fragments were subjected to crystal trials in the presence of single-stranded DNA, and diffraction quality crystals were obtained for RPA181-422 bound to octadeoxycytidine. The RPA181-422 co-crystals belonged to the P2(1)2(1)2(1) space group, with unit cell dimensions of a = 34.3 A, b = 78.0 A, and c = 95.4 A and diffracted to a resolution of 2.1 A.

Crystallography, X-Ray↗
PubMed1996

Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein, EBNA1, bound to DNA.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds to and activates DNA replication from oriP, the latent origin of DNA replication in Epstein-Barr virus. The crystal structure of the DNA-binding domain of EBNA1 bound to an 18 bp binding site was solved at 2.4 A resolution. EBNA1 comprises two domains, a flanking and a core domain. The flanking domain, which includes a helix that projects into the major groove and an extended chain that travels along the minor groove, makes all of the sequence-determining contacts with the DNA. The core domain, which is structurally homologous to the complete DNA-binding domain of the bovine papilloma virus E2 protein, makes no direct contacts with the DNA bases. A model for origin unwinding is proposed that incorporates the known biochemical and structural features of the EBNA1-origin interaction.

Amino Acid Sequence↗
PubMed1995

Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein EBNA 1.

The crystal structure of the DNA-binding and dimerization domains of the Epstein-Barr virus nuclear antigen 1 (EBNA1), which binds to and activates DNA replication from the latent origin of replication in Epstein-Barr virus, was solved at 2.5 A resolution. EBNA1 appears to bind DNA via two independent regions termed the core and the flanking DNA-binding domains. The core DNA-binding domain, which comprises both the dimerization domain and a helix predicted to bind the inner portion of the EBNA1 DNA recognition element, was remarkably similar to the structure of the papillomavirus E2 protein, despite a complete lack of sequence conservation. The flanking DNA-binding domain, only a portion of which is contained in the current structure, consists in part of an alpha helix whose N-terminus contacts the outer regions of the EBNA1 DNA recognition element.

Amino Acid Sequence↗
PubMed1995

Overexpression, purification, and crystallization of the DNA binding and dimerization domains of the Epstein-Barr virus nuclear antigen 1.

The Epstein-Barr virus nuclear antigen (EBNA) 1 binds to and activates DNA replication from the latent origin of Epstein-Barr virus. Six different fragments of EBNA1 that retain DNA binding activity were expressed in bacteria, purified, and crystallized. Two fragments, EBNA470-619 and EBNA470-607, formed well ordered crystals that diffracted beyond 2.5-A resolution. Two different EBNA470-619 crystals were grown from sodium formate, pH 6-6.5. One crystal belonged to the trigonal space group P3 with unit cell dimensions a = b = 86.5 A and c = 31.8 A and with two molecules in the asymmetric unit. The other crystal, which appeared only twice and was likely related to the P3 crystal form, belonged to the trigonal space group P312 with cell dimensions a = b = 86.7 A and c = 31.8 A. Crystals of EBNA470-607 were grown by lowering the salt concentration to 0-100 mM NaCl at pH 6.0. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and had cell dimensions a = 59 A, b = 66.9 A, and c = 69.8 A with two molecules in the asymmetric unit.

Antigens, Viral↗
PubMed1995

The mechanism of protein crystal growth from lipid layers.

Two-dimensional (2D) crystals of proteins on lipid monolayers can initiate the formation of large three-dimensional (3D) crystals suitable for X-ray diffraction studies. The role of the 2D crystals in this process has not been firmly established. While it is likely that the 2D crystals serve as nuclei for epitaxial crystal growth, other mechanisms, such as non-specific nucleation induced by the high local concentration of the protein at the surface of the lipid layer, cannot be excluded. Using streptavidin as a model system, we have now firmly established that 3D crystal growth from 2D crystals on lipid layers occurs by epitaxy. We show that 2D crystals of streptavidin (space group C222) on biotinated lipid layers nucleate the growth of a 3D crystal form (space group I4I22) that possesses a structural similarity with the 2D crystal, but have no effect on the growth of 3D crystal forms (I222 and P2(1)) that are unrelated to the 2D crystal. At lower pH, a new 3D crystal form (space group P1), unrelated to the previously described 2D crystals, grew from lipid layers. This discovery initially raised concern about the validity of the epitaxial mechanism, but these concerns were alleviated with the subsequent discovery of a structurally related 2D P1 crystal that grew in similar solution conditions. Some parameters affecting epitaxial growth of both the P1 and I4I22 crystals were investigated, revealing several noteworthy features of the epitaxial growth. (1) 2D crystals are very effective nucleating agents; for instance, the P1 2D crystals can direct the growth of P1 3D crystals even under conditions that favour the growth of other crystal forms. (2) The epitaxial 3D crystal grow very rapidly and at amazingly low protein concentrations; P1 3D crystals can be grown from solutions as low as 10 microM streptavidin. (3) There is no obligate requirement for the deposition of pre-formed 2D crystals; lipid layers alone are equally effective at promoting epitaxial crystal growth.

Bacterial Proteins↗