DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.     

Role of papillomavirus E1 initiator dimerization in DNA replication

Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.     

Alleviation of restriction by DNA condensation and non-specific DNA binding ligands

During conditions of cell stress, the type I restriction and modification enzymes of bacteria show reduced, but not zero, levels of restriction of unmethylated foreign DNA. In such conditions, chemically identical unmethylated recognition sequences also occur on the chromosome of the host but restriction alleviation prevents the enzymes from destroying the host DNA. How is this distinction between chemically identical DNA molecules achieved? For some, but not all, type I restriction enzymes, alleviation is partially due to proteolytic degradation of a subunit of the enzyme. We identify that the additional alleviation factor is attributable to the structural difference between foreign DNA entering the cell as a random coil and host DNA, which exists in a condensed nucleoid structure coated with many non-specific ligands. The type I restriction enzyme is able to destroy the ‘naked’ DNA using a complex reaction linked to DNA translocation, but this essential translocation process is inhibited by DNA condensation and the presence of non-specific ligands bound along the DNA.     

Region-specific DNA damage by AT-specific DNA-reactive drugs is predicted by drug binding specificity

Bizelesin and adozelesin are DNA-reactive antitumor drugs that alkylate adenines at the 3' ends of their preferred binding sites [5'T(A/T)(4)A3'and 5'(A/T)(3)(-4)A3', respectively]. We used these drugs to examine the determinants for region-specific damage of human genomic DNA. The distribution of bizelesin binding motifs in several regions analyzed "in silico" correlated well with the experimentally determined lesions in these regions assessed by quantitative polymerase chain reaction (QPCR) stop assay. In contrast to the typically low motif density, clusters of potential bizelesin binding sites were found in the matrix-associated regions (MAR domains) of the c-myc and apolipoprotein B (apoB) genes. Accordingly, lesions induced by bizelesin in these domains (2.13 and 7.06 lesions kbp(-1) microM(-1), respectively) markedly exceeded lesions in bulk DNA (0.87 lesions kbp(-1) microM(-1)) or in regions with typically low motif density (e.g., 0.75 and 0.87 lesions kbp(-1) microM(-1) in a beta-globin gene and c-myc origin of replication regions, respectively). Consistent with the more frequent, less localized adozelesin motif, actual lesions induced by adozelesin exceeded by severalfold lesions by bizelesin in four selected regions (within the c-myc and HPRT loci). Whereas adozelesin is likely to affect similar regions as bizelesin, adozelesin's more promiscuous binding probably compromises its relative specificity for such targets. In contrast, findings for bizelesin provide for the first time a proof of principle that a small molecular weight drug can preferentially damage specific regions in cellular DNA. Targeting of critical repetitive sequences, such as AT-rich MAR domains, which allow for clustering of drug binding motif, can be the paradigm for region specificity of small molecular weight agents.     

Mechanism of inhibition of DNA gyrase by quinolone antibacterials: specificity and cooperativity of drug binding to DNA

Although the functional target of quinolone antibacterials such as nalidixic acid and norfloxacin has been identified as the enzyme DNA gyrase, the direct binding site of the drug is the DNA molecule [Shen, L. L., & Pernet, A. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 307-311]. As described in this paper, binding specificity and cooperativity of quinolones to DNA were further investigated with the use of a variety of DNA species of different structures and different base compositions. Results show that the drug binding specificity is controlled and determined largely by the DNA structure. The drug binds weakly and demonstrates no base preference when DNA strands are paired. The drug binds with much greater affinity when the strands are separated, and consequently, binding preference emerges: it binds better to poly(G) and poly(dG) over their counterparts including poly(dI). The results suggest that the drug binds to unpaired bases via hydrogen bonding and not via ring stacking with DNA bases. The weak binding to relaxed double-stranded DNA and the stronger binding to single-stranded DNA are both nonspecific as they do not demonstrate binding saturation and cooperativity. The specific type of binding, initially demonstrated in our previous publication with the supercoiled DNA and more recently with complex formed between linear DNA and DNA gyrase [Shen, L. L., Kohlbrenner, W. E., Weigl, D., & Baranowski, J. (1989) J. Biol. Chem. (in press)], occurs near the drug's supercoiling inhibition concentration. As shown in this paper, binding saturation curves of this type are highly cooperative (with Hill constant greater than 4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Intradomain LexA rotation is a prerequisite for DNA binding specificity

In the absence of DNA damage the LexA protein represses the bacterial SOS system. We performed molecular dynamic simulations of two LexA dimers bound to operators. Our model predicted that rotation of the LexA DNA binding domain, with respect to the dimerised C-terminal domain, is required for selective DNA binding. To confirm the model, double and quadruple cysteine LexA mutants were engineered. Electrophoretic mobility-shift assay and surface plasmon resonance showed that disulfide bond formation between the introduced cysteine residues precluded LexA specific DNA binding due to blocked domain reorientation. Our model could provide the basis for novel drug design.     

ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the glucocorticoid receptor

The 70-kDa heat shock protein (Hsp70) is involved in providing the appropriate conformation of various nuclear hormone receptors, including the glucocorticoid receptor (GR). The Bcl-2 associated athanogene 1M (Bag-1M) is known to downregulate the DNA binding by the GR. Also, Bag-1M interacts with the ATPase domain of Hsp70 to modulate the release of the substrate from Hsp70. In this study, we demonstrate that ATP hydrolysis enhances Bag-1M-mediated inhibition of the DNA binding by the GR. However, the inhibitory effect of Bag-1M was abolished when the intracellular ATP was depleted. In addition, a Bag-1M mutant lacking the interaction with Hsp70 did not influence the GR to bind DNA, suggesting the interaction of Bag-1M with Hsp70 in needed for its negative effect. These results indicate that ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the GR and Hsp70 is a mediator for this process.     

DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.

Bovine papillomavirus type 1 (BPV-1) DNA replicates episomally and requires two virally expressed proteins, E1 and E2, for this process. Both proteins bind to the BPV-1 genome in the region that functions as the origin of replication. The binding sequences for the E2 protein have been characterized previously, but little is known about critical sequence requirements for E1 binding. Using a bacterially expressed E1 fusion protein, we examined binding of the BPV-1 E1 protein to the origin region. E1 strongly protected a 28-bp segment of the origin (nucleotides 7932 to 15) from both DNase I and exonuclease III digestion. Additional exonuclease III protection was observed beyond the core region on both the 5' and 3' sides, suggesting that E1 interacted with more distal sequences as well. Within the 28-bp protected core, there were two overlapping imperfect inverted repeats (IR), one of 27 bp and one of 18 bp. We show that sequences within the smaller, 18-bp IR element were sufficient for specific recognition of DNA by E1 and that additional BPV-1 sequences beyond the 18-bp IR element did not significantly increase origin binding by E1 protein. While the 18-bp IR element contained sequences sufficient for specific binding by E1, E1 did not form a stable complex with just the isolated 18-bp element. Formation of a detectable E1-DNA complex required that the 18-bp IR be flanked by additional DNA sequences. Furthermore, binding of E1 to DNA containing the 18-bp IR increased as a function of overall increasing fragment length. We conclude that E1-DNA interactions outside the boundaries of the 18-bp IR are important for thermodynamic stabilization of the E1-DNA complex. However, since the flanking sequences need not be derived from BPV-1, these distal E1-DNA interactions are not sequence specific. Comparison of the 18-bp IR from BPV-1 with the corresponding region from other papillomaviruses revealed a symmetric conserved consensus sequence, T-RY--TTAA--RY-A, that may reflect the specific nucleotides critical for E1-DNA recognition.     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of the sequence non-specific DNA binding HMG1/2-box family of proteins, has been examined by both mutation analyses and molecular modeling techniques. Substitution of the residue 102F, which is characteristically exposed to solvent, with a small hydrophobic amino acid affected its DNA binding activity. However, no additional effect was observed by the further mutation of flanking 101F. Molecular dynamics simulation and modeling studies revealed that 102F intercalates into DNA base-pairs, being supported by the flanking 101F. The mutants with a small hydrophobic residue at position 102 tolerated the substitution for 101F because the side chain at position 102 is too short to intercalate. Thus the intercalation of 102F and the positive effect of the flanking 101F residue are important for the sequence non-specific DNA binding of the HMG1/2-box. The conserved basic residues of 95K, 96R and 109R were also examined for their roles in DNA binding. These residues interacted with DNA mainly by electrostatic interaction and maintained the location of the box on the DNA, which prescribed the intercalation of 102F. The DNA intercalation by HMG1 consists of an ingenious mechanism which brings DNA conformational changes necessary for biological functions.     

The molecular basis of selective DNA binding by the BRG1 AT-hook and bromodomain

The ATP-dependent BAF chromatin remodeling complex plays a critical role in gene regulation by modulating chromatin architecture, and is frequently mutated in cancer. Indeed, subunits of the BAF complex are found to be mutated in >20% of human tumors. The mechanism by which BAF properly navigates chromatin is not fully understood, but is thought to involve a multivalent network of histone and DNA contacts. We previously identified a composite domain in the BRG1 ATPase subunit that is capable of associating with both histones and DNA in a multivalent manner. Mapping the DNA binding pocket revealed that it contains several cancer mutations. Here, we utilize SELEX-seq to investigate the DNA specificity of this composite domain and NMR spectroscopy and molecular modelling to determine the structural basis of DNA binding. Finally, we demonstrate that cancer mutations in this domain alter the mode of DNA association.     

The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs) on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3), hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.     

a1 protein alters the DNA binding specificity of alpha 2 repressor

The alpha 2 protein of S. cerevisiae, the product of the MAT alpha 2 gene, represses a set of cell-type-specific genes (the a-specific genes) by binding to an operator sequence upstream of each gene. We demonstrate that a second yeast regulatory protein, a1, the product of the MATa1 gene, can alter the binding specificity of alpha 2 so that it no longer recognizes the a-specific gene operator, but instead acquires the ability to recognize a different operator sequence found upstream of haploid-specific genes. Thus, under the influence of a1, alpha 2 can repress haploid-specific genes. An alpha cell expresses alpha 2 but not a1, so that alpha 2 turns off only the a-specific genes. An a/alpha cell makes both a1 and alpha 2, in a ratio that ensures that alpha 2 is distributed between two distinct binding modes: the alpha 2 binding mode and the a1-alpha 2 binding mode. Thus in an a/alpha cell, alpha 2 represses two distinct classes of genes.     

The E1 initiator recognizes multiple overlapping sites in the papillomavirus origin of DNA replication

A common feature of replicator sequences from a variety of organisms is multiple binding sites for an initiator protein. By binding to the replicator, initiators mark the site and contribute to melting or distortion of the DNA. We have defined the recognition sequence for the papillomavirus E1 initiator and determined the arrangement of binding sites in the viral origin of replication. We show that E1 recognizes a hexanucleotide sequence which is present in overlapping arrays in virtually all papillomavirus replicators. Binding of the initiator to these sites would result in the formation of a closely packed array of E1 molecules that wrap around the double helix.