Distamycin and penta-N-methylpyrrolecarboxamide binding sites on native DNA. A comparison of methidiumpropyl-EDTA-Fe(II) footprinting and DNA affinity cleaving

Using two direct methods we have studied the binding locations and site sizes of distamycin and penta-N-methylpyrrolecarboxamide on three DNA restriction fragments from pBR322 plasmid. We find that methidiumpropyl-EDTA.Fe(II) footprinting and DNA affinity cleaving methods report common binding locations and site sizes for the tri- and pentapeptides bound to heterogeneous DNA. The tripeptide distamycin binds 5-base-pair sites with a preference for poly(dA).poly(dT) regions. The pentapeptide binds 6-7-base-pair sites with a preference for poly(dA).poly(dT) regions. These results are consistent with distamycin binding as an isogeometric helix to the minor groove of DNA with the four carboxamide N-H's hydrogen bonding five A + T base pairs. The data supports a model where each of the carboxamide N-H's can hydrogen bond to two bases, either O(2) of thymine or N(3) of adenine, located on adjacent base pairs on opposite strands of the helix. In most (but not all) cases the tri- and pentapeptide can adopt two orientations at each A + T rich binding site.     

基于进化足迹法结合位置打分函数的转录因子结合位点预测

目的:预测转录因子结合位点。方法:本文从ABS数据库上下载了人类和啮齿类动物的直系同源的启动子序列,首先使用位置打分函数对这些启动子序列进行打分,找到候选的转录因子结合位点,并进一步利用进化足迹法对这些候选的转录因子结合位点进行筛选,只有结合位点同时出现在人类和啮齿类动物的启动子序列才认为是真正的结合位点。结果:在对人类和啮齿类动物进行转录因子结合位点预测时,与单独使用打分函数的结果相比,进化足迹法与打分函数结合的方法有效的提高了预测结果的性能,大幅度提供所得预测结果的特异性。结论:进化足迹法结合位置打分矩阵的方法能较为准确有效的预测转录因子结合位点。     

DNA足纹步移作图法

一种以PCR介导的、可对任意长度靶DNA片段上的核蛋白结合位点进行DNA足纹作图分析的新方法.原理是：采用被随机降解靶DNA分子作为模板,用标记的跨越整个模板的足够多条特异性引物进行单链扩增.首先,利用某种化学试剂或酶如DNaseⅠ对已与蛋白质结合或未结合的双链靶DNA进行随机降解,在一定条件下使每一个DNA分子恰好只有一个位点被切割.然后,这些被随机降解DNA分子即可作为模板,用同位素标记的跨越整个模板的足够多条特异性引物（正向或反向）进行单链扩增.最后,扩增的单链产物通过变性聚丙烯酰胺凝胶电泳和放射自显影形成DNA片段梯队,而被蛋白质保护的位点则在DNA片段梯队中形成位置缺口,从而确定DNA与蛋白质相结合的精确位点.该方法被应用对人干细胞因子基因5′旁侧－1190～－273区域的DNA足纹部分作图.     

An Unexpected Major Groove Binding of Netropsin and Distamycin A to tRNAphe

Abstract

Crystalline complexes of yeast tRNA^phe and the oligopeptide antibiotics netropsin and distamycin A were prepared by diffusing drugs into crystals of tRNA. X-ray structure analyses of these complexes reveal a single common binding site for both drugs which is located in the major or deep groove of the tRNA T-stem. The netropsin-tRNA complex is stabilized by specific hydrogen bonds between the amide groups of the drug and the tRNA bases G51 0(6), U52 0(4) and G53 N(7) on one strand, and is further stabilized by electrostatic interactions between the positively charges guanidino side chain of the drug and the tRNA phosphate P53 on the same strand and the positively charged amidino propyl side chain and the phosphates P61, P62 and P63 on the opposite strand of the double helix. These results are in contrast to the implicated minor groove binding of these drugs to non-guanine sequences in DNA. The binding to the GUG sequence in tRNA implies that major groove binding to certain DNA sequences is possible.     

Steric Block High Affinity Oligonucleotide Analogues: A New Tool for Mapping RNA-Protein Binding Sites

Steric-block ON analogues are efficient inhibitors of RNA-protein interaction and therefore have potential to probe RNA sequences for putative protein binding sites and to investigate mechanisms of protein binding. The packaging process of HIV-1 is highly specific involving an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. Using oligonucleotide probes we have confirmed that the terminal purine loop is the major Gag binding site on SL3 and that a secondary Gag binding site exists at an internal purine bulge. We also demonstrate direct binding of oligonucleotide to their binding sites and confirm this interaction does not alter global RNA conformation, making them highly specific, nondisruptive probes of RNA protein interactions.     

Effect of DNA Local Conformation on the Affinity and Binding Specificity of bis-Netropsins to DNA

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked tail-to-tail via cis-diammineplatinum group (Nt–Pt(NH ₃ )–Nt) or aliphatic pentamethylene chain (Nt–(CH ₂ ) ₅ –Nt), has been studied. Both bis-netropsins have been shown to bind to DNA oligomer 5"-CCTATATCC-3" (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5"-CCXCC-3" [where X = TTATT (II), TTAT (III), TTTTT (IV), and AATTT (V)] along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of Nt–(CH ₂ ) ₅ –Nt with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin ( ₂) to monodentate binding constant ( ₁) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for Nt–Pt(NH ₃ )–Nt and Nt–(CH ₂ ) ₅ –Nt binding as a hairpin has been found to be DNA duplex 5"-CGTATACG-3".     

Affinity Labeling of Adenosine A1 Binding Sites

The adenosine A1 receptors of sheep brain membranes have been identified by the specific binding of radiolabeled cyclohexyl[3H]adenosine ([3H]CHA). Pretreatment of membranes with periodate-oxidized CHA causes a dose- and time-dependent decrease in the number of binding sites. No decrease occurs when membranes are pretreated with CHA. Binding of [3H]CHA to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.     

Defining the DNA Substrate Binding Sites on HIV-1 Integrase

A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3′ processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3′ processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench.     

Poly(U)-Agarose Affinity Chromatography: Specific,Sensitivity Selectivity,and Affinity of Binding

These studie s were done to determine four basic intrinsic properties of poly(U)-agarose affinity columns. Specificity of binding studies demonstrated that binding to these columns is highly specific with >90% complementary binding and 3% noncomplementary binding. Sensitivity of binding studies indicated that a minimum sequence of 10 adenylates is required for detectable complementary binding. Selectivity of binding studies revealed that nonsequential adenylates in native RNAs and randomly distribut edadenylates in synthetic poly(A)-poly(C) co-polymers did not bind to poly(U)-agarose affinity columns. Whereas, affinity of binding studies demonstrated that A=U complementary base pairing is independent of chain-lengths of 25 a denylates and dependent of chain-lengths of <25 adenylates. Thus the data demonstrates that poly(U)-agarose affinity chromatography is scientifically sound and expedient for thedetection and isolation of poly(A)-containing cellular and viral RNAs.     

Evidence that Protein Binding Specifies Sites of DNA Demethylation

It has been hypothesized that protein factors may protect CpG islands from methyltransferase during development and that demethylation may involve protein-DNA interactions at demethylated sites. However, direct evidence has been lacking. In this study, demethylation at the EBNA-1 binding sites of the Epstein-Barr virus latent replication origin, oriP, was investigated by using human cells. Several novel findings are discussed. First, there are specific preferential demethylation sites within the oriP region. Second, the DNA sequence of oriP alone is not the target of an active demethylation process. Third, EBNA-1 binding is required for the site-specific demethylation in oriP. Interestingly, CpG sites adjacent to and between the EBNA-1 sites do not become demethylated. Fourth, demethylation of the first DNA strand in oriP at the EBNA-1 binding sites involves a passive (replication-dependent) mechanism. The second-strand demethylation appears to occur through an active mechanism. That is, EBNA-1 protein binding prevents the EBNA-1 binding sites from being remethylated after one round of DNA replication, and it appears that an active demethylase then demethylates these hemimethylated sites. This study provides clear evidence that protein binding specifies sites of DNA demethylation and provides insights into the sequence of steps and the mechanism of demethylation.     

Structure and Dynamics of Distamycin A with d(CGCAAATTGGC):d(GCCAATTTGCG) at Low Drug: DNA Ratios

Abstract

Two-dimensional NMR has been used to study the interaction of distamycin A with d(CGCAAA- TTGGC):d(GCCAATTTGCG) at low and intermediate drug: DNA ratios (<2.0). Drug-DNA contacts were identified by nuclear Overhauser effect spectroscopy, which also served to monitor exchange of the drug between different binding sites. At low drug: DNA ratios (0.5), distamycin A binds in two orientations within the five central A-T base pairs and has a preference (2.2:1) for binding with the formyl end directed toward the 5′ side of the A-rich strand. The pattern of drug-DNA contacts corresponding to the preferred binding orientation are consistent with the drug sliding between adjacent AAAT and AATT binding sites at a rate that is fast on the NMR time scale. Similarly, the pattern of NOEs associated with the less favored orientation are consistent with the drug sliding between adjacent AATT and ATTT sites, again in fast exchange. Off-rates for the drug from the major and minor binding orientations were measured to be 2.4 =1.5 and 3.3 = 1.5 s^?1 respectively, at 35°C. At intermediate drug: DNA ratios (1.3) exchange of the drug between the two one-drug and the two sites of a two-drug complex is observed. Off-rates for both drugs from the 2:1 complex were measured to be 1.0 =0.5 s^?1 (35°C).     

Signatures of Protein-DNA Recognition in Free DNA Binding Sites

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel β strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A-B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein-DNA interactions and thus lend insights towards a structural code for DNA recognition.