首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 312 毫秒
1.
2.
Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.  相似文献   

3.
A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.  相似文献   

4.
The human T-cell leukemia virus Tax protein directs binding of a host factor, cAMP response element binding protein, to an extended recognition sequence in the proviral promoter. Prior cross-linking experiments have revealed that Tax makes restricted contact with this DNA at two symmetric positions, 14 nucleotides apart on opposite strands of the DNA. Tax lacks a conventional DNA binding domain, and the sequences in Tax that are in contact with DNA have not been previously identified. Analysis of cross-linked peptides now shows that the contact occurs between Tax residues 89 and 110, corresponding to a protease-sensitive linker joining two protein structural domains. The linker assumes a protease-resistant conformation in the cross-linked complex. Point mutations within the linker prevent cross-linking and interfere with Tax function. These data suggest that entry of Tax into the ternary complex may be coupled to folding of an unstructured protein domain, which then makes base-specific contacts with DNA.  相似文献   

5.
Cross-linking studies with the uvrA and uvrB proteins of E. coli   总被引:3,自引:0,他引:3  
P Strike  W D Rupp 《Mutation research》1985,145(1-2):43-48
The interactions of the uvrA and uvrB proteins with DNA have been investigated using a DNA-protein cross-linking technique. It is demonstrated that hydrolysis of ATP by the uvrA protein facilitates cross-linking of this protein to single-stranded DNA, whether the DNA is UV irradiated or not. In contrast, cross-linking to unirradiated double-stranded DNA is not facilitated by ATP hydrolysis and is in fact increased by the substitution of the non-hydrolysable analogue aTP gamma S for ATP. In the presence of ATP, a dose-dependent increase is observed in the amount of uvrA protein which can be cross-linked to UV-irradiated double-stranded DNA. Binding of uvrB protein to puvrA-DNA complexes has a stabilising effect and increases the number of complexes which can be cross-linked whether the substrate is single- or double-stranded DNA. We can find no evidence that ATP hydrolysis by uvrA protein results in unwinding of UV-damaged DNA.  相似文献   

6.
The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif.  相似文献   

7.
To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.  相似文献   

8.
Human nucleotide excision repair is initiated by six repair factors (XPA, RPA, XPC-HR23B, TFIIH, XPF-ERCC1, and XPG) which sequentially assemble at sites of DNA damage and effect excision of damage-containing oligonucleotides. We here describe the molecular anatomy of the human excision nuclease assembled at the site of a psoralen-adducted thymine. Three polypeptides, primarily positioned 5' to the damage, are in close physical proximity to the psoralen lesion and thus are cross-linked to the damaged DNA: these proteins are RPA70, RPA32, and the XPD subunit of TFIIH. While both XPA and XPC bind damaged DNA and are required for XPD cross-linking to the psoralen-adducted base, neither XPA nor XPC is cross-linked to the psoralen adduct. The presence of other repair factors, in particular TFIIH, alters the mode of RPA binding and the position of its subunits relative to the psoralen lesion. Based on these results, we propose that RPA70 makes the initial contact with psoralen-damaged DNA but that within preincision complexes, it is RPA32 and XPD that are in close contact with the lesion.  相似文献   

9.
V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.  相似文献   

10.
This study attempted to characterize proteins cross-linked to DNA of Yoshida lymphosarcoma cells treated with methylene dimethanesulfonate (MDMS) and its hydrolytic products formaldehyde (HCHO) and methanesulfonic acid (MSA). MDMS and HCHO treatments produced a similar extent and type of DNA-protein cross-linking in Yoshida lymphosarcoma cells. All five major histones (H1, H2a, H2b, H3, and H4) were among the nuclear proteins cross-linked to DNA. Certain discrete differences were also apparent in these studies. MDMS cross-linked proteins of 29 and 48 kDa to DNA that were not observed following HCHO treatment alone, and HCHO cross-linked a 26-kDa protein to DNA that was not observed following MDMS treatment. Because semicarbazide prevented all MDMS-induced DNA-protein cross-linking, HCHO must be the component responsible for this lesion. The 26-kDa protein has been identified as an H4-H2b dimer. The formation of this dimer is particularly sensitive to MSA release on hydrolysis of MDMS because, in the presence of MSA, HCHO preferentially cross-linked an H2a-H2b dimer and a 48-kDa non-histone protein to DNA. Differences in DNA-protein cross-linking between these two agents are therefore proposed to arise from discrete changes in chromatin structure induced directly by MSA release.  相似文献   

11.
12.
A E Grebanier  A O Pogo 《Cell》1979,18(4):1091-1099
Reversible cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells was used as a probe to determine whether the protein structure was preserved following treatment with DNAase I. Interactions between histones were analyzed through cross-linking with 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate. No alterations in the interactions between intranucleosomal histone proteins resulted from digestion of the nuclear DNA. There was, however, a diminished extent of cross-linking of histone H1 to itself and to the intranucleosomal histones in DNA-depleted nuclei. The interactions of a group of nonhistone proteins with histone H3 could be monitored by cross-linking through the formation of disulfide bonds caused by oxidation of nuclei with H2O2. These interactions were not markedly affected by treatment of the nuclei with DNAase I. However, differences were observed in the extent of cross-linking of some of these proteins when cross-linking in nuclei from undifferentiated cells was compared to that in nuclei from cells which had been induced to differentiate with dimethylsulfoxide.  相似文献   

13.
14.
PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA. Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI.  相似文献   

15.
Strand-specific binding of RPA and XPA to damaged duplex DNA   总被引:7,自引:0,他引:7  
The nucleotide excision repair (NER) pathway is a major pathway used to repair bulky adduct DNA damage. Two proteins, xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA), have been implicated in the role of DNA damage recognition in the NER pathway. The particular manner in which these two damage recognition proteins align themselves with respect to a damaged DNA site was assessed using photoreactive base analogues within specific DNA substrates to allow site-specific cross-linking of the damage recognition proteins. Results of these studies demonstrate that both RPA and XPA are in close proximity to the adduct as measured by cross-linking of each protein directly to the platinum moiety. Additional studies demonstrate that XPA contacts both the damaged and undamaged strands of the duplex DNA. Direct evidence is presented demonstrating preferential binding of RPA to the undamaged strand of a duplex damaged DNA molecule.  相似文献   

16.
A highly photosensitive analogue of thymidine, 5-azidodeoxyuridine 5'-triphosphate, has been incorporated into 61-base pair (bp) DNA fragments corresponding to the central region of Xenopus somatic-type 5 S RNA genes such that 5-azidodeoxyuridine replaces some or all T residues in either the coding or noncoding strand of the TFIIIA binding site. Photolysis of TFIIIA.DNA complexes formed with these probes results in efficient, sequence-specific cross-linking to the Zn-finger protein providing direct evidence that this class of proteins have contacts in the major groove of their target sequence. Of the 20 T residues present in the 61-bp probes, greater than 90% of the cross-linking occurs from two sites in the 5 S RNA gene corresponding to T residues at positions 84 and 88 in the noncoding and coding strands, respectively. Digestion by V8 protease of the complex formed with the noncoding strand probe releases peptides not bound to the DNA. Amino acid sequence analysis of the remaining, cross-linked peptides indicates the region including zinc-finger 2 plus the finger 2-3 linker is in contact with position 84. The linker region between fingers 5 and 6 is also in close proximity to the major groove somewhere upstream from position 84.  相似文献   

17.
An essential step in the development of the vertebrate immune system is the DNA level rearrangement of the antigen receptor genes. This process, termed "V(D)J recombination," begins with DNA cleavage at the appropriate sites mediated by the two proteins RAG1 and RAG2. We report here that the two proteins cooperate to bind DNA with significantly higher specificity than either protein alone. Gel purification of the triple complex is performed in the absence of any cross-linking agents. Both proteins remain present in the complex, and UV cross-linking using iodouridine-containing probes shows that RAG1 makes close contacts in both the heptamer and nonamer motifs. The two proteins are also shown to associate with each other in the absence of any DNA. These findings refine our understanding of the protein-DNA interactions that accompany cleavage at the recombination signals.  相似文献   

18.
19.
Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号