Preferential binding to DNA sequences of peptides related to a novel XPRK motif

Two dodecapeptide amines: (WPRK)(3)NH(2)[WR-12] and (YPRK)(3)NH(2)[YR-12], and a 30-mer polypeptide amide (SP-30) were synthesized by solid-phase peptide methodology. DNase I footprinting studies on a 117-mer DNA showed that WR-12 and YR-12 bind selectively to DNA sequences in a manner similar to SP-30 which has a repeating SPK(R)K sequence. The most distinctive blockages seen with all three peptides occur at positions 26-30, 21-24 and 38-45 around sequences 5'-GAATT-3', 5'-TAAT-3' and 5'-AAAACGAC-3', respectively. However, it appears that YR-12 is better able to extend its recognition site to include CG pairs than is SP-30. At low concentrations YR-12 was able to induce enhanced rates of DNase I cleavage at regions surrounding some of its binding sites. To obtain further quantitative data supplementary to the footprinting work, equilibrium binding experiments were performed in which the binding of the two peptides to six decanucleotide duplexes was compared. Scatchard analyses indicated that WR-12 may be more selective for oligomers containing runs of consecutive purines or pyrimidines. On the other hand, YR-12 binds better to d(CTTAGACGTC)- d(GACGTCTAAG) than to the other oligomer duplexes, denoting selectivity for evenly distributed C/G and A/T sequences.     

Properties of DNase I digestion of the deoxyoligonucleotide: 5''d(ATCGTACGAT)2(3'').

Deoxyribonuclease I digestion of the deoxyoligodecamer 5'd(ATCGTACGAT)2(3') has been examined in detail to study the kinetic and structural properties of this enzyme substrate system in solution. In addition, these studies have defined, in general, those DNase I conditions to be used in future drug-DNA footprinting experiments. Special attention has been taken of those properties of DNase I that are critical for quantitation of ligand binding to small DNA fragments, and that aid in designing oligomers to be used in footprinting experiments. Enzyme activity was observed at all phosphodiester bonds in the decamer studied with varying affinity, except for the first four bonds at the 5' end of the oligomer. The DNA substrate concentration is always in excess, in order to achieve conditions of no more than one DNase I cleavage per DNA molecule. Reactions were controlled so that 65% or more of the initial amount of decamer substrate remained after DNase I digestion. It was observed that the rate of enzyme reactivity decreases with digestion time and is sensitive to the experimental conditions.     

Characterization of major recognition sequences for a herpes simplex virus type 1 origin-binding protein.

To investigate early initiation events in the replication of herpes simplex virus type 1, we analyzed interactions of proteins from infected cell extracts with the small origin of herpes simplex virus type 1 (oris1). Using the mobility shift assay, we detected two origin-specific binding interactions. We characterized the more prominent interaction on both strands of the DNA duplex with DNase I protection and methylation interference assays. Protein binding protects 17 bases of DNA on each strand from DNase I. These sequences are located at the left end of the central palindrome and are shifted four bases relative to one another. On the basis of the DNase protection pattern, we believe this protein to be related to the origin-binding protein defined by Elias et al. (P. Elias, M.E. O'Donnell, E.S. Mocarski, and I.R. Lehman, Proc. Natl. Acad. Sci. 83:6322-6326, 1986). Our DNase I footprint shows both strong and weak areas of protection. The regions strongly protected from DNase I align with the essential contact residues identified by interference footprinting. Methylation interference defines a small binding domain of 8 base pairs: 5'-GTTCGCAC-3'/3'-CAAGCGTG-5'. This recognition sequence contains two inverted 5'-GT(T/G)CG-3' repeats which share a 2-base overlap; thus, the origin-binding protein probably binds to the inverted repeats as a dimer.     

鼠疫菌cAMP受体蛋白对毒力相关基因yopD调控的初步研究

目的研究鼠疫菌cAMP受体蛋白（CRP）对毒力相关基因yopD的调控情况。方法利用芯片表达谱和实时定量RT-PCR初步判断CRP对yopD的调控,在获得纯化的his-CRP蛋白后进行体外凝胶阻滞实验（EMSA）证明CRP能否直接结合yopD启动子区,DNase I足迹实验（DNase I footprinting assay）确定yopD启动子区CRP结合位点序列精确序列。结果芯片表达谱和实时定量RT-PCR实验一致证实CRP可能直接负调控yopD,EMSA证明CRP能直接结合yopD启动子区,DNase I足迹实验确定yopD的启动子区上CRP位点的精确序列。结论CRP可能直接负调控毒力相关基因仰D。     

Binding of f-PIP, a pyrrole- and imidazole-containing triamide, to the inverted CCAAT box-2 of the topoisomerase IIalpha promoter and modulation of gene expression in cells

An N-formamido pyrrole- and imidazole-containing triamide (f-PIP) has been shown by DNase I footprinting, SPR, and CD studies to bind as a stacked dimer to its cognate sequences: 5'-TACGAT-3' (5'-flank of the inverted CCAAT box-2 of the human topoisomerase IIalpha promoter) and 5'-ATCGAT-3'. A gel shift experiment provided evidence for f-PIP to inhibit protein-DNA interaction at the ICB2 site. Western blot studies showed that expression of the topoisomerase IIalpha gene in confluent NIH 3T3 cells was induced by treatment with f-PIP. The results suggested that the triamide was able to enter the nucleus, interacted with the target site within ICB2, inhibited NF-Y binding, and activated gene expression.     

Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter.

Genomic footprinting on the Drosophila hsp26 promoter in isolated nuclei has shown that a TATA box binding factor is present before and after induction by heat shock, while three of the seven heat shock consensus sequences 5' of the gene are occupied (presumably by heat shock factor, HSF) specifically on heat shock. The sites of HSF interaction are separated by greater than 200 bp of which approximately 150 bp are bound to the surface of a nucleosome. The juxtaposition of these various macromolecules on the DNA suggests a basis for the major DNase I hypersensitive site 5' of hsp26 and a novel tertiary structure for the promoter complex.     

Synthesis and biophysical evaluation of minor-groove binding C-terminus modified pyrrole and imidazole triamide analogs of distamycin

Five polyamide derivatives with rationally modified C-terminus moieties were synthesized and their DNA binding specificity and affinity determined. A convergent approach was employed to synthesize polyamides containing an alkylaminopiperazine (4 and 5), a truncated piperazine (6), or an alkyldiamino-C-terminus moiety (7 and 8) with two specific objectives: to investigate the effects of number of potential cationic centers and steric bulk at the C-terminus. CD studies confirmed that compounds 4, 5, 7, and 8 bind in the minor groove of DNA. The alkylpiperazine containing compounds (4 and 5) showed only moderate binding to DNA with DeltaT(m) values of 2.8 and 8.3 degrees C with their cognate sequence, respectively. The alkyldiamino compounds (7 and 8) were more impressive producing a DeltaT(m) of >17 and >22 degrees C, respectively. Compound 6 (truncated piperazine) did not stabilize its cognate DNA sequence. Footprints were observed for all compounds (except compound 6) with their cognate DNA sequence using DNase I footprinting, with compound 7 producing a footprint of 0.1 microM at the expected 5'-ACGCGT-3' site. SPR analysis of compound 7 binding to 5'-ACGCGT-3', 5'-ACCGGT-3', and 5'-AAATTT-3' produced binding affinities of 2.2x10(6), 3.3x10(5), and 1x10(5)M(-1), respectively, indicating a preference for its cognate sequence of 5'-ACGCGT-3'. These results are in good agreement with the footprinting data. The results indicate that steric crowding at the C-terminus is important with respect to binding. However, the number of cationic centers within the molecule may also play a role. The alkyldiamino-containing compounds (7 and 8) warrant further investigation in the field of polyamide research.     

A Streptomyces glaucescens endodeoxyribonuclease which shows a strong preference for CC dinucleotide.

We have characterized a deoxyribonuclease from Streptomyces glaucescens that cleaves double-stranded DNA preferably between the dinucleotide 5'-CC-3'. The cleavage specificity was demonstrated by both analysis of the terminal nucleotides of the generated fragments and DNA sequencing of partially digested DNA. Digestion of lambda DNA with this enzyme resulted in the production of double-stranded fragments with 5' and/or 3'-protruding single-stranded tails. DNase I footprinting experiments indicated that the nuclease specifically binds to its cleavage sites on the DNA under non-catalytic conditions. The enzyme is not affected by cytosine methylation in hemimethylated DNA.     

Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5-thyminyl-5,6-dihydrothymine) but not cyclobutane pyrimidine dimers in UV-irradiated DNA

The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the splB gene in Bacillus subtilis. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant B. subtilis spores and shown to efficiently cleave SP but not cyclobutane cis,syn thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an Escherichia coli overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py lozengePy) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py lozengePy-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the (32)P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3' to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.     

Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S. cerevisiae.

Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and Werner's syndrome genes. We have studied the substrate specificity of a recombinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein). Sgs1 shows a strong preference for binding branched DNA substrates, including duplex structures with a 3' single-stranded overhang and DNA junctions with multiple branches. Duplex DNA with a 5' rather than a 3' single-stranded tail is not recognized or unwound by Sgs1. DNase I and hydroxyl radical footprinting of the Sgs1-DNA complex shows that the protein binds specifically to the junction of a double-stranded DNA and its 3' overhang. Binding and unwinding of duplex DNA with a 3' overhang are much reduced if the backbone polarity of the 3' overhang is reversed in the junction region, but are unaffected if polarity reversal occurs four nucleotides away from the junction. These results indicate that the 3' to 5' polarity of unwinding by the recombinant Sgs1 protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinant Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo.     

Quantitative footprinting analysis of the chromomycin A3--DNA interaction.

Chromomycin A3 (CHR) binding to the duplex d(CAAGTCTGGCCATCAGTC).d(GACTGATGGCCAGACTTG) has been studied using quantitative footprinting methods. Previous NMR studies indicated CHR binds as a dimer in the minor groove. Analysis of autoradiographic spot intensities derived from DNase I cleavage of the 18-mer in the presence of various amounts of CHR revealed that the drug binds as a dimer to the sequence 5'-TGGCCA-3',3'-ACCGGT-5' in the 18-mer with a binding constant of (2.7 +/- 1.4) x 10(7) M-1. Footprinting and fluorescence data indicate that the dimerization constant for the drug in solution is approximately 10(5) M-1. Since it has been suggested that CHR binding alters DNA to the A configuration, quantitative footprinting studies using dimethyl sulfate, which alkylates at N-7 of guanine in the major groove, were also carried out. Apparently, any drug-induced alteration in DNA structure does not affect cleavage by DMS enough to be observed by these experiments.     

DNA sequence-specific recognition of peptides incorporating the HPRK and polyamide motifs

Three peptide amides, HPRK(Py)(4)HPRK-NH(2) (PyH-12), HPRK(Py)(3)HPRK-NH(2) (PyH-11) and HPRK(Py)(2)HPRK-NH(2) (PyH-10), incorporating two HPRK motifs and various 4-amino-1-methylpyrrole-2-carboxylic acid residues (Py) were synthesized by solid-phase peptide methodology. The binding of these three peptides to a 5'-32P-labeled 158-mer DNA duplex (Watson fragment) and to a 5'-32P-labeled 135-mer DNA duplex (complementary Crick fragment) was investigated by quantitative DNase I footprinting. On the 158-mer Watson strand, the most distinctive DNase I blockages seen with all three peptides occur around positions 105-112 and 76-79, corresponding to the sequences 5'-GAGAAAAT-3' and 5'-CGGT-3', respectively. However, on the complementary Crick strand, only PyH-12 strongly discriminates the 5'-TTT-3' site around positions 108-110 whereas both PyH-11 and PyH-10 have moderate binding around positions 102-112 comprising the sequence 5'-ATTTTCTCCTT-3'. Possible bidentate and single interactions of the side-chain functions and alpha-amino protons of the peptides with DNA bases are discussed.     

Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5'-ACGCGT-3'

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5'-ACGCGT-3'. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔT(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5'-ACGCGT-3'. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 10(7)M(-1), which is ～3 times higher than that for its monoamino analog 3 (4.8 × 10(6)M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2.     

Methidiumpropyl-EDTA.Fe(II) and DNase I footprinting report different small molecule binding site sizes on DNA.

DNase I and MPE.Fe (II) footprinting both employ partial cleavage of ligand-protected DNA restriction fragments and Maxam-Gilbert sequencing gel methods of analysis. One method utilizes the enzyme, DNase I, as the DNA cleaving agent while the other employs the synthetic molecule, methidium-propyl-EDTA (MPE). For actinomycin D, chromomycin A3 and distamycin A, DNase I footprinting reports larger binding site sizes than MPE.Fe (II). DNase I footprinting appears more sensitive for weakly bound sites. MPE.Fe (II) footprinting appears more accurate in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA. MPE.Fe (II) and DNase I report the same sequence and binding site size for lac repressor protein on operator DNA.     

Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs.

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.     

Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.