Identification of functionally important residues in the DNA binding region of the mnt repressor

Single amino acid substitutions have been introduced throughout the N-terminal DNA binding region of the Mnt repressor, and the operator binding properties of the resulting mutant repressors have been assayed. These studies show that the side chains of Arg2, His6, Asn8, and Arg10 are critical for high affinity binding to operator DNA. Other side chains in the N-terminal region do not appear to play major roles in DNA recognition and binding. Specific alterations in the pattern of methylation protection afforded by the Arg2----Lys mutant protein suggest that Arg2 contacts the N7 groups of guanines 10 and 12 in the operator. In conjunction with previous results, these findings suggest that part of the N-terminal region of Mnt binds as an extended polypeptide strand within the major groove of the mnt operator.     

Local sequence of protein β‐strands influences twist and bend angles

β‐Sheet twisting is thought to be mainly determined by interstrand hydrogen bonds with little contribution from side chains, but some proteins have large, flat β‐sheets, suggesting that side chains influence β‐structures. We therefore investigated the relationship between amino acid composition and twists or bends of β‐strands. We calculated and statistically analyzed the twist and bend angles of short frames of β‐strands in known protein structures. The most frequent twist angles were strongly negatively correlated with the proportion of hydrophilic amino acid residues. The majority of hydrophilic residues (except serine and threonine) were found in the edge regions of β‐strands, suggesting that the side chains of these residues likely do not affect β‐strand structure. In contrast, the majority of serine, threonine, and asparagine side‐chains in β‐strands made contacts with a nitrogen atom of the main chain, suggesting that these residues suppress β‐strand twisting. Proteins 2014; 82:1484–1493. © 2014 Wiley Periodicals, Inc.     

Random coil structures in bacterial proteins. Relationships of their amino acid compositions to flanking structures and corresponding genic base compositions

In this study we classified regions of random coil into four types: coil between alpha helix and beta strand, coil between beta strand and alpha helix, coil between two alpha helices and coil between two beta strands. This classification may be considered as natural. We used 610 3D structures of proteins collected from the Protein Data Bank from bacteria with low, average and high genomic GC-content. Relatively short regions of coil are not random: certain amino acid residues are more or less frequent in each of the types of coil. Namely, hydrophobic amino acids with branched side chains (Ile, Val and Leu) are rare in coil between two beta strands, unlike some acrophilic amino acids (Asp, Asn and Gly). In contrast, coil between two alpha helices is enriched by Leu. Regions of coil between alpha helix and beta strand are enriched by positively charged amino acids (Arg and Lys), while the usage of residues with side chains possessing hydroxyl group (Ser and Thr) is low in them, in contrast to the regions of coil between beta strand and alpha helix. Regions of coil between beta strand and alpha helix are significantly enriched by Cys residues. The response to the symmetric mutational pressure (AT-pressure or GC-pressure) is also quite different for four types of coil. The most conserved regions of coil are “connecting bridges” between beta strand and alpha helix, since their amino acid content shows less strong dependence on GC-content of genes than amino acid contents of other three types of coil. Possible causes and consequences of the described differences in amino acid content distribution between different types of random coil have been discussed.     

Preferred interaction of d-peptidyl-anthraquinones with double-stranded B-DNA

The quest for more specific drugs in antitumor chemotherapy led us to the design of anthraquinone–peptide conjugates capable of selective recognition of the nucleic acid. We present here the DNA binding characteristics, sequence specificity and geometry of interaction of a pair of enantiomers containing the lysine–glycine dipeptide in the side chains. The d enantiomer binds right handed double stranded DNA more efficiently than the l form under all conditions tested. The source of higher binding affinity is not electrostatic in nature and rests in the more favorable hydrophobic contacts of the d-lysyl side chains in the drug-DNA complex. Both derivatives exhibit preference for alternating GC base sequences and intercalate into DNA in a threading mode as suggested by chiroptical and theoretical studies. The d enantiomer, being a peptidyl derivative that contains a non-natural amino acid, has the considerable advantage of being less susceptible to enzymatic hydrolysis and could therefore represent a lead compound for further development.     

Systematic Analysis of Possible Hydrogen Bonds between Amino Acid Side Chains and B-form DNA

Abstract

The ways in which amino acid side chains could make a pair of hydrogen bonds within the major groove of B DNA are systematically analyzed. Hydrogen bond donors within the major groove are characterized by determining the idealized position of the hydrogen bond acceptors that they might bond with. It appears that an amino acid side chain could, at most, contact a pair of base pairs. The ten possible pairs of base pairs are analyzed to determine how they could be recognized by the amino acid side chains.     

Synthesis and properties of novel psoralen derivatives

We have synthesized a set of new trimethylpsoralen derivatives that are characterized by a chain extending from the 4'-position of the furan ring and linked to this ring by an aminomethylene group. The nature of the side chain can be varied widely. In these derivatives, the chains contain either amino or ethylene oxide units for enhanced water solubility and allow the introduction of a thiol or amine group to nucleic acids. These compounds represent the first set of thiolated psoralen derivatives, and their usefulness is demonstrated in several nucleic acid cross-linking experiments. The reagents can be used to create both intraduplex reversible cross-links between the two single-strand partners in a DNA double helix and interduplex reversible cross-links between two DNA double helices.     

Binding of DNA to large fragment of DNA polymerase I: identification of strong and weak electrostatic forces and their biological implications.

Examination of the electrostatic potential of a modeled complex, consisting of the Klenow fragment of E. coli DNA polymerase I and DNA template-primer, suggested the presence of two distinct interacting regions. The one displaying a strong electropositive potential field is generated by side chains of basic amino acid pairs and is directed towards the major groove site in DNA. The second electrostatic potential field around DNA is somewhat weaker and appears to be exerted by a pair of vicinal side chains of acidic and basic amino acids. The distribution of charges in this manner appears well suited for the binding of enzyme to the template-primer required in the enzymatic synthesis of DNA.     

Novel peptide derivatives of bleomycin A5: synthesis, antitumor activity and interaction with DNA

A series of novel amino acid and peptide derivatives of bleomycin (BLM) A(5) were synthesized. All the compounds possessed significant antitumor activities in vitro against HL-60, BGC-823, PC-3MIE8, and MDA-MB-435 cell lines. Their antitumor activities against MDA-MB-435 were 10-fold higher than BLM A5. The DNA cleavage studies indicated that the hydrophobic amino acid or peptide derivatives of BLM A5 could induce higher cleavage ratio of double to single strand DNA than BLM A5. From the DNA binding studies, we found that the derivatives containing either D-conformation amino acid or basic amino acid could facilitate DNA binding of BLM.     

Structural insight into the substrate specificity of DNA Polymerase mu

DNA polymerase mu (Pol mu) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol mu's unusual substrate specificity, we describe the 2.4 A crystal structure of the polymerase domain of murine Pol mu bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol mu that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol mu from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3' ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.     

Ned Seeman and the prediction of amino acid-basepair motifs mediating protein-nucleic acid recognition

Fifty years ago, the first atomic-resolution structure of a nucleic acid double helix, the mini-duplex (ApU)₂, revealed details of basepair geometry, stacking, sugar conformation, and backbone torsion angles, thereby superseding earlier models based on x-ray fiber diffraction, including the original DNA double helix proposed by Watson and Crick. Just 3 years later, in 1976, Ned Seeman, John Rosenberg, and Alex Rich leapt from their structures of mini-duplexes and H-bonding motifs between bases in small-molecule structures and transfer RNA to predicting how proteins could sequence specifically recognize double helix nucleic acids. They proposed interactions between amino acid side chains and nucleobases mediated by two hydrogen bonds in the major or minor grooves. One of these, the arginine-guanine pair, emerged as the most favored amino acid-base interaction in experimental structures of protein-nucleic acid complexes determined since 1986. In this brief review we revisit the pioneering work by Seeman et al. and discuss the importance of the arginine-guanine pairing motif.     

Two helix DNA binding motif of CAP found in lac repressor and gal repressor.

Comparison of both the DNA and protein sequences of catabolite gene activator protein (CAP) with the sequences of lac and gal repressors shows significant homologies between a sequence that forms a two alpha-helix motif in CAP and sequences near the amino terminus of both repressors. This two-helix motif is thought to be involved in specific DNA sequence recognition by CAP. The region in lac repressor to which CAP is homologous contains many i-d mutations that are defective in DNA binding. Less significant sequence homologies between CAP and phage repressors and activators are also shown. The amino acid residues that are critical to the formation of the two-helix motif are conserved, while those residues expected to interact with DNA are variable. These observations suggest the lac and gal repressors also have a two alpha-helix structural motif which is involved in DNA binding and that this two helix motif may be generally found in many bacterial and phage repressors. We conclude that one major mechanism by which proteins can recognize specific base sequences in double stranded DNA is via the amino acid side chains of alpha-helices fitting into the major groove of B-DNA.     

Analysis of the beta-delta-globin gene loci in normal and Hb Lepore DNA: direct determination of gene linkage and intergene distance

Total human DNA was cleaved with a variety of restriction enzymes, and the fragments were fractionated by gel electrophoresis and transferred to nitrocellulose filter strips. The restricted DNA was then hybridized to nick-translated radioactive recombinant plasmid DNA containing sequences derived from human beta-globin messenger RNA. Under suitable conditions, this probe hybridizes with both the beta--and delta-globin genes. Using this probe, a restriction map of the human beta--and delta-globin genes and the surrounding genomic DNA regions has been constructed. The beta-globin gene contains a nonglobin DNA insert approximately 899-1000 base pairs in length, present within the sequence coding for amino acids 101-120 of the 146 amino acid long globin polypeptide. A similar sequence may be present within the same sequence of the delta-globin gene. The distance between the beta--and delta-globin genes is approximately 7000 nucleotide pairs, and the delta-globin gene is to the 5' side of the beta-globin gene, as predicted by genetic evidence. Both genes are transcribed from the same DNA strand. The structure of the Hb Lepore gene is shown to be a fused delta--and beta-globin gene, and to be completely consistent with the derived map of normal beta--and delta-globin genes. [Restriction enzyme nomenclature follows that of Smith and Nathans (1973) and Roberts (1976). A genomic DNA restriction fragment containing part or all of one globin gene will be designated by that globin chain--for instance, the Pst I fragment containing the beta-globin gene sequence will be designated Pst I beta. A similar convention will be used for double digests. Throughout this paper, when reference is made to the 5' or 3' side or fragment of a gene, this refers to the 5' or 3' side of the mRNA coded by that sequence. Thus the 5' side (N terminal) of the beta-globulin gene is the sequence to the 5' side of the anti-sense strand.].     

Induction of DNA double strand breaks in cultured mammalian cells exposed to hydrogen peroxide and histidine

The amino acid histidine was found to increase the toxicity of H₂O₂ in cultured mammalian cells. Histidine also augmented the level of DNA single strand breaks (SSB) detectable in cells exposed to the oxidant and, in addition, resulted in the appearance of DNA double strand breaks (DSB), a lesion which is not produced by H₂O₂ alone.     

Crystal structure of the first KH domain of human poly(C)-binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1.7 A

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-A resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.     

Elimination of double strand nuclease activity from S1 nuclease prepared from crude alpha amylase.

Single strand-specific s1 nuclease prepared as previously described from crude alpha amylase by DEAE-cellulose chromatography also contains nuclease which degrades double strand nucleic acid. The double strand activity can be removed by repeating the DEAE-cellulose chromatography procedure at least two additional times. S1 nuclease prepared by this procedure does not degrade double strand sheared DNA as measured by Sephadex chromatography. Under the same conditions single strand DNA is completely degraded. Thus, S1 nuclease prepared by this procedure is suitable for use in removing single strand regions in DNA/DNA duplexes and DNA/RNA hybrids.     

Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria

The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.     

Promoting DNA molecules association by amphiphilic derivatives of 1,3-diazaadamantanes containing hydrophobic side chains

Earlier, a new class of compounds, amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides, has been discovered. Longer hydrophobic side chains in 1,3-diazaadamantanes have been found to promote stronger acceleration of the reaction. In this study, the interaction of two 1,3-diazaadamantane derivatives containing different side chains with DNA was investigated using optical methods. Concentrations of micelle formation by the 1,3-diazaadamantanes, as well as the ranges of concentrations where the compounds/water mixtures exist in the form of true solutions, were determined based on the increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate probe. The affinities of 1,3-diazaadamantanes to DNA were determined with fluorescent intercalator displacement (FID) assay. A significant increase in the hydrodynamic volume of short DNA hairpins in complexes with 1,3-diazaadamantanes was revealed by the estimation of the fluorescence polarization of ethidium bromide probe bound in the hairpins. The intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantanes was confirmed by Förster resonance energy transfer in an equimolar mixture of hairpins fluorescently labeled with Cy-3 or Cy5. In the study, the number of positive charges on 1,3-diazaadamantane derivatives that contain side chains of different lengths was demonstrated to affect their affinity to DNA, while longer hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate DNA strand exchange.     

The actions of Neurospora endo-exonuclease on double strand DNAs

Neurospora crassa endo-exonuclease, an enzyme implicated in recombinational DNA repair, was found previously to have a distributive endonuclease activity with a high specificity for single strand DNA and a highly processive exonuclease activity. The activities of endo-exonuclease on double strand DNA substrates have been further explored. Endo-exonuclease was shown to have a low bona fide endonuclease activity with completely relaxed covalently closed circular DNA and made site-specific breaks in linear double strand DNA at a low frequency while simultaneously generating a relatively high level of single strand breaks (nicks) in the DNA. Sequencing at nicks induced by endo-exonuclease in pBR322 restriction fragments showed that the highest frequency of nicking occurred at the mid-points of two sites with the common sequence, p-AGCACT-OH. In addition, sequencing revealed less frequent nicking at identical or homologous hexanucleotide sequences in all other 54 cases examined where these sequences either straddled the break site itself or were within a few nucleotides on either side of the break site. The exonucleolytic action of endo-exonuclease on linear DNA showed about 100-fold preference for acting in the 5' to 3' direction. Removal of the 5'-terminal phosphates substantially reduced this activity, internal nicking, and the ability of endo-exonuclease to generate site-specific double strand breaks. On the other hand, nicking of the dephosphorylated double strand DNA with pancreatic DNase I stimulated the exonuclease activity by almost 5-fold, but no stimulation was observed when the DNA was nicked by Micrococcal nuclease. Thus, 5'-p termini either at double strand ends or at nicks in double strand DNA are entry points to the duplex from which endo-exonuclease diffuses linearly or "tracks" in the 5' to 3' direction to initiate its major endo- and exonucleolytic actions. The results are interpreted to show how it is possible for endo-exonuclease to generate single strand DNA for switching into a homologous duplex either at a nick or while remaining bound at a double strand break in the DNA. Such mechanisms are consistent with current models for recombinational double strand break repair in eukaryotes.     

Construction of nonnatural DNA library.

Nonnatural DNA polymerase substrates which contain many kinds of modified functional groups are synthesized. The C-5 position of dUTP was modified by amino acid, saccharide. These compounds were incorporated on a DNA double strand using E. Coli DNA polymerase. This nonnatural DNA library contains a larger diversity than native DNA or RNA and has a higher chemical stability than RNA. This library will be useful for in vitro selection study, combinatorial chemistry, and the preparation of supramolecular structures.