Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000.

Plasmid pAL5000 from Mycobacterium fortuitum encodes two proteins necessary for replication: RepA (307 amino acid residues) and RepB (119 residues). A single RNA species encoding these proteins was characterized, and its 5' end was defined. The proteins were expressed as maltose-binding protein fusions in Escherichia coli. The RepB protein was shown in vitro to bind specifically to a previously defined 435-bp region of pAL5000 containing the origin of replication (ori). The precise RepB binding sites were defined by DNase I footprinting experiments. RepB binds to two motifs in the ori region: a high-affinity site within its own promoter region, implying autoregulation of its expression, and a low-affinity site further upstream, presumably the origin of replication itself. The binding to the latter motif seems to occur on one DNA strand only. The high-affinity binding site contains several palindromic sequences. Gel retardation assays were performed with the different binding sites as templates, and the binding constant to each site was estimated from protein titrations. This is the first molecular dissection of mycobacterial DNA-binding proteins and their interactions with their targets.     

SPKK, a new nucleic acid-binding unit of protein found in histone.

A new DNA-binding unit of a protein different from the alpha-helix, the beta-sheet and the Zn-finger is proposed based on the analysis of the structure of the N-terminus of sea urchin spermatogenous histone H1. DNA-binding arms of the sea urchin spermatogenous histones, H1 and H2B, are composed of repeats of Ser-Pro-Lys(Arg)-Lys(Arg) (SPKK) residues. A six-times repeat of SPKK (S6 peptide) was isolated from H1 and the competition of S6 for DNA binding with a DNA-binding dye, Hoechst 33258, was analysed. The S6 peptide is shown to be a competitive inhibitor of Hoechst 33258, and it is concluded that the SPKK repeat binds to DNA in its minor groove with a binding constant, KS6 = 1.67 X 10(10) M-1. The circular dichroism (CD) spectrum of a synthetic peptide, SPRKSPRK (S2 peptide), is quite different from those of both the alpha-helix and the beta-sheet and resembles that of a random coil. From statistical consideration of protein structures it is proposed that SPKK forms a compact beta-turn stabilized by an additional hydrogen bond. Since a repeated chain of such turn of SPKK offers a repeat of amides of Ser residues at a distance similar to that of DNA-binding amides of the drugs, Hoechst 33258 and netropsin, and since the amides of these drugs bind to DNA replacing the spine of hydration in a minor groove, it is proposed that a repeat of SPKK binds to DNA in the minor groove using similar hydrogen bonds.     

Study of the ATP-binding site of helicase IV from Escherichia coli

Helicases contain conserved motifs involved in ATP/magnesium/nucleic acid binding and in the mechanisms coupling nucleotide hydrolysis to duplex unwinding. None of these motifs are located at the adenine-binding pocket of the protein. We show here that the superfamily I helicase, helicase IV from Escherichia coli, utilizes a conserved glutamine and conserved aromatic residue to interact with ATP. Other superfamily I helicases such as, UvrD/Rep/PcrA also possess these residues but in addition they interact with adenine via a conserved arginine, which is replaced by a serine in helicase IV. Mutation of this serine residue in helicase IV into histidine or methionine leads to proteins with unaffected ATPase and DNA-binding activities but with low helicase activity. This suggests that residues located at the adenine-binding pocket, in addition to be involved in ATP-binding, are important for efficient coupling between ATP hydrolysis and DNA unwinding.     

A protease-sensitive hinge linking the two domains of the hepatitis B virus core protein is exposed on the viral capsid surface.

Core particles of hepatitis B virus are assembled from dimers of a single 185-residue (subtype adw) viral capsid or core protein (p21.5) which possesses two distinct domains: residues 1 to 144 form a minimal capsid assembly domain, and the arginine-rich, carboxyl-terminal residues 150 to 185 form a protamine-like domain that mediates nucleic acid binding. Little is known about the topography of the p21.5 polypeptide within either the p21.5 capsids or dimers. Here, using site-specific proteases and monoclonal antibodies, we have defined the accessibility of p21.5 residues in dimers and capsids assembled from wild-type and mutant hepatitis B virus core proteins in Xenopus oocytes and in vitro. The data reveal the protamine region to be accessible to external reagents in p21.5 dimers but largely cryptic in wild-type capsids. Strikingly, in capsids the only protease target region was a 9-residue peptide covering p21.5 residues Glu-145 to Asp-153, which falls largely between the two core protein domains. By analogy with protease-sensitive interdomain regions in other proteins, we propose that this peptide constitutes a hinge between the assembly and nucleic acid binding domains of p21.5. We further found that deletion or replacement of the terminal Cys-185 residue greatly increased surface exposure of the protamine tails in capsids, suggesting that a known disulfide linkage involving this residue tethers the protamine region inside the core particles. We propose that disruption of this disulfide linkage allows the protamine region to appear transiently on the surface of the core particle.     

Interaction of human papillomavirus (HPV) type 16 capsid proteins with HPV DNA requires an intact L2 N-terminal sequence.

Encapsidation of papillomavirus DNA involves DNA-protein and protein-protein interactions. We sought to define the role of each human papillomavirus (HPV) capsid protein in HPV DNA encapsidation. HPV16 major (L1) and minor (L2) capsid proteins purified from recombinant vaccinia virus-infected cells were compared for their ability to bind nucleic acids. L2 protein, but not L1 protein, could bind HPV DNA. To map the DNA-binding region of L2, a series of truncated or point-mutated L2 protein open reading frames were used to show that only the N terminal of L2 was required for L2-DNA binding. This interaction depends critically on charged amino acids (Lys or Arg) in the first 12 amino acids of the N terminal of the protein. Several techniques were used to show that L2 interaction with DNA did not require specific DNA sequences. We propose that HPV L2 protein may play a major role in papillomavirus capsid assembly by introducing HPV DNA to the virus particles formed by the self assembly of the L1 major structural protein.     

Nucleotide sequence and role in DNA amplification of the direct repeats composing the amplifiable element AUD1 of Streptomyces lividans 66

The amplifiable unit of DNA no. 1 (AUD1) of Streptomyces lividans consists of three 1 kb repeats (left direct repeat, LDR; middle direct repeat, MDR; and the slightly different right direct repeat, RDR) and two 4.7 kb repeats alternately arranged in identical orientation to each other. Both 4.7 kb repeats have been sequenced. They are identical and contain one open reading frame ( orf4.7  ). The deduced amino acid sequence has a low similarity to chitinases, and two amino acid repeats present high similarities to fibronectin type III modules. Sequencing had previously shown that the ORF corresponding to each 1 kb repeat encodes a putative DNA-binding protein. Crude extracts of Escherichia coli overexpressing the orfRDR- encoded protein and of S. lividans Jni1, having a high amplification of AUD1 and therefore orfMDR , were used in gel retardation assays. The orfRDR - and probably the orfMDR -encoded proteins can bind to an imperfect palindromic sequence upstream from MDR and RDR and to another sequence downstream from RDR. An extrachromosomal DNA amplification system was constructed containing different combinations of the sequences composing AUD1. In mutants having a deletion of the chromosomal AUD1, the 4.7 kb repeats could be reduced in size, mutated or replaced by E. coli DNA without altering the ability to amplify when RDR was present. Therefore, the only function of the 4.7 kb repeats in amplification is to provide directly repeated DNA sequences. When RDR was lacking or mutated, no amplification was observed. This strongly suggests that the DNA-binding protein encoded by orfRDR is required for AUD1 amplification.     

A plant gene encoding a Myb-like protein that binds telomeric GGTTTAG repeats in vitro

A gene (AtTRP1) encoding a telomeric repeat-binding protein has been isolated from Arabidopsis thaliana. AtTRP1 is a single copy gene located on chromosome 5 of A. thaliana. The protein AtTRP1 encoded by this gene is not only homologous to the Myb DNA-binding motifs of other telomere-binding proteins but also is similar to several initiator-binding proteins in plants. Gel retardation assay revealed that the 115 residues on the C terminus of this protein, including the Myb motif, are sufficient for binding to the double-stranded plant telomeric sequence. The isolated DNA-binding domain of AtTRP1 recognizes each telomeric repeat centered on the sequence GGTTTAG. The almost full-length protein of AtTRP1 does not form any complex at all with the DNA fragments carrying four or fewer GGTTTAG repeats. However, it forms a complex with the sequence (GGTTTAG)(8) more efficiently than with the sequence (GGTTTAG)(5). These data suggest that the minimum length of a telomeric DNA for AtTRP1 binding consists of five GGTTTAG repeats and that the optimal AtTRP1 binding may require eight or more GGTTTAG repeats. It also implies that this protein AtTRP1 may bind in vivo primarily to the ends of plant chromosomes, which consist of long stretches of telomeric repeats.     

Interaction of the IciA protein with AT-rich regions in plasmid replication origins.

A set of AT-rich repeats is a common motif in prokaryotic replication origins. We have screened for proteins binding to the AT-rich repeat region in plasmids F, R1 and pSC101 using an electrophoretic mobility shift assay with PCR-amplified DNA fragments from the origins. The IciA protein, which is known to bind to the AT-rich repeat region in the Escherichia coli origin of chromosome replication, oriC, was found to bind to the corresponding region from plasmids F (oriS) and R1, but not to pSC101. DNase I footprint analysis showed that IciA interacted with the AT-rich region in both F and R1. When the IciA gene was deleted, the copy number of plasmid F increased somewhat, whereas there was no major effect on the replication of pSC101 and R1, or on the E. coli chromosome.     

Inhibition of Epstein-Barr virus OriP function by tankyrase, a telomere-associated poly-ADP ribose polymerase that binds and modifies EBNA1

Tankyrase (TNKS) is a telomere-associated poly-ADP ribose polymerase (PARP) that has been implicated along with several telomere repeat binding factors in the regulation of Epstein-Barr virus origin of plasmid replication (OriP). We now show that TNKS1 can bind to the family of repeats (FR) and dyad symmetry regions of OriP by using a chromatin immunoprecipitation assay and DNA affinity purification. TNKS1 and TNKS2 bound to EBNA1 in coimmunoprecipitation experiments with transfected cell lysates and with purified recombinant proteins in vitro. Two RXXPDG-like TNKS-interacting motifs in the EBNA1 amino-terminal domain mediated binding with the ankyrin repeat domain of TNKS. Mutations of both motifs at EBNA1 G81 and G425 abrogated TNKS binding and enhanced EBNA1-dependent replication of OriP. Small hairpin RNA targeted knock-down of TNKS1 enhanced OriP-dependent DNA replication. Overexpression of TNKS1 or TNKS2 inhibited OriP-dependent DNA replication, while a PARP-inactive form of TNKS2 (M1045V) was compromised for this inhibition. We show that EBNA1 is subject to PAR modification in vivo and to TNKS1-mediated PAR modification in vitro. These results indicate that TNKS proteins can interact directly with the EBNA1 protein, associate with the FR region of OriP in vivo, and inhibit OriP replication in a PARP-dependent manner.     

Pentatricopeptide repeat proteins and their emerging roles in plants.

Several protein families with tandem repeat motifs play a very important role in plant development and defense. The pentatricopeptide repeat (PPR) protein family, one of the largest families, is the most perplexing one in plants. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. PPR motifs are degenerate motifs, each with 35-amino-acid sequences and are present in tandem arrays of 2-27 repeats per protein. Although PPR proteins are found in other eukaryotes, their large number is probably required in plants to meet the specific needs of organellar gene expression. The repeats of PPR proteins form a superhelical structure to bind a specific ligand, probably a single-stranded RNA molecule, and modulate its expression. Functional studies on different PPR proteins have revealed their role in organellar RNA processing, fertility restoration in CMS plants, embryogenesis, and plant development. Functional genomic techniques can help identify the diverse roles of the PPR family of proteins in nucleus-organelle interaction and in plant development.     

Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein

Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely alpha-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys(216) and Tyr(217), binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.