Interaction between UV-damaged DNA binding activity proteins and the c-Abl tyrosine kinase

The c-Abl tyrosine kinase is activated by some forms of DNA damage, including ionizing radiation, but not UV radiation. The functions of this activation in the damage response pathways remain obscure. To identify potential targets of c-Abl kinase, we utilized the yeast two-hybrid system to screen a murine cDNA library. One c-Abl binding protein of particular interest was the large subunit (DDB1) of the heterodimeric complex with UV-damaged DNA binding activity (UV-DDB). This complex binds with high specificity to DNA damaged by UV, is absent in a subset of xeroderma pigmentosum group E cells, and is required for global genomic repair of UV-induced damage. The association of c-Abl with DDB1 required the kinase domain of c-Abl and preserved the interaction between DDB1 and the small subunit (DDB2) of the UV-DDB complex. Significantly, overexpression of c-Abl increased tyrosine phosphorylation of DDB2 and suppressed UV-DDB activity. Conversely, a dominant negative, kinase-deficient allele of c-Abl decreased tyrosine phosphorylation of DDB2 and dramatically stimulated UV-DDB activity. These results suggest that one role of c-Abl may be to negatively regulate UV-DDB activity by phosphorylation of DDB2.     

Characterization of distamycin A binding to damaged DNA

We previously reported that distamycin A, a natural antibiotic known as a minor groove binder, could bind to DNA duplexes containing the (6-4) photoproduct formed at its target site, whereas the binding was not observed for duplexes containing the cis-syn cyclobutane pyrimidine dimer in the same sequence context. In this study, we have further analyzed the binding of this drug to lesion-containing duplexes to elucidate its damaged-DNA recognition mechanism. Surface plasmon resonance measurements using various types of DNA showed that distamycin A could bind to several types of lesion-containing DNA. Curve fitting of the CD titration data revealed that the complex formation occurred with K(d) values around 10(-6) and a stoichiometry of 1:1. The results obtained in this study suggested that distamycin A binds to damaged DNA in the same way as to the normal target site, by recognizing the chemical structure of the minor groove.     

A DNA binding protein from human placenta specific for ultraviolet damaged DNA.

A DNA-binding protein specific for ultraviolet irradiated DNA has been purified extensively from human placenta. The binding preparation is free of exonuclease, polymerase, endonuclease, and N-glycosidase activity. The binding activity is salt dependent and is specific for double-stranded irradiated DNA. DNA from which the pyrimidine dimers have been monomerized by the action of photolyase (photoreactivating enzyme) remains an effective substrate for the binding protein, suggesting that the protein recognizes photoproducts other than pyrimidine dimers. This is supported by the finding that DNA irradiated under conditions which introduce only pyrimidine dimers is not a substrate for the binding protein. Examination of three of the xeroderma pigmentosum complementation groups has revealed no deficiency in this binding activity.     

Recognition of damaged regions in DNA by oligopeptides and proteins

The binding of various damaged DNAs to the single-strand binding protein coded for by gene 32 from bacteriophage T4, on the one hand, and of oligopeptides containing tryptophan and lysine residues, on the other hand, is described. These molecules exhibit a higher affinity for modified DNA than for native DNA in so far as modification results in a local destabilization of the double-stranded structure of the nucleic acid. Stacking interactions between aromatic amino acids and nucleic acid bases appear to play a crucial role in the recognition of destabilized regions induced by chemical agents (carcinogens and antitumor drugs). These interactions confer to the peptide lysyl-tryptophyl-lysine an endonucleolytic activity specific for apurinic sites. From results obtained with such oligopeptides a model for the active sites of Ap-endonucleases is proposed which could account for the strategy used by the denV endonuclease from phage T4 during the first step of excision repair of pyrimidine dimers in DNA. The effect of the overall conformation of modified DNA on repair efficiency is discussed.     

A kinase-independent function of Ask1 in caspase-independent cell death

Ask1 (apoptosis signal-regulating kinase 1) is activated as a consequence of cell exposure to a variety of stresses and can then initiate apoptosis. A known pathway of apoptosis downstream of Ask1 involves the activation of the stress-activated protein kinases, the release of cytochrome c from mitochondria, the activation of caspases, and the fragmentation of nuclei. Here, we characterized a novel mechanism of Ask1-mediated cell killing that is triggered by the interaction with Daxx. Co-transfection of Ask1 and Daxx induced a caspase-independent cell-death process characterized at the morphological level by distinctive crumpled nuclei easily distinguishable from the condensed and fragmented nuclei seen during classical caspase-dependent apoptosis. The kinase activity of Ask1 was not involved in this process, because mutants lacking kinase activity were as efficient as wild type Ask1 in mediating Daxx-induced cell death. Ask1N, a deletant that lacks the C-terminal half including the kinase domain of Ask1, was constitutively active in producing crumpled nuclei. In contrast, Ask1DeltaN, the reciprocal deletant that possesses constitutive kinase activity, produced fragmented nuclei typical of caspase-dependent death processes. We conclude that in addition to a caspase-dependent pro-apoptotic function that depends on its kinase activity, Ask1 possesses a caspase-independent killing function that is independent on its kinase activity and is activable by interaction with Daxx. In the physiological situation, such an activity is induced as a consequence of the translocation of Daxx from the nucleus to the cytoplasm, a condition that occurs following activation of the death receptor Fas.     

Using structural motif templates to identify proteins with DNA binding function

This work describes a method for predicting DNA binding function from structure using 3-dimensional templates. Proteins that bind DNA using small contiguous helix–turn–helix (HTH) motifs comprise a significant number of all DNA-binding proteins. A structural template library of seven HTH motifs has been created from non-homologous DNA-binding proteins in the Protein Data Bank. The templates were used to scan complete protein structures using an algorithm that calculated the root mean squared deviation (rmsd) for the optimal superposition of each template on each structure, based on C_α backbone coordinates. Distributions of rmsd values for known HTH-containing proteins (true hits) and non-HTH proteins (false hits) were calculated. A threshold value of 1.6 Å rmsd was selected that gave a true hit rate of 88.4% and a false positive rate of 0.7%. The false positive rate was further reduced to 0.5% by introducing an accessible surface area threshold value of 990 Ų per HTH motif. The template library and the validated thresholds were used to make predictions for target proteins from a structural genomics project.     

c-Abl在DNA损伤反应中的功能

非受体酪氨酸激酶c-Abl在正常生理及病理条件下具有多种生物学功能。当电离辐射、顺铂、丝裂霉素C等DNA损伤诱导剂诱导DNA损伤反应后,c-Abl可参与DNA损伤反应后的细胞周期调控、基因重组修复及细胞凋亡调控等,进而决定细胞在DNA损伤反应条件下的状态。简要介绍了c-Abl在DNA损伤反应中的作用及其进展。     

Strand-specific binding of RPA and XPA to damaged duplex DNA

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.     

Effects of DNA binding proteins on DNA methylation in vitro

The inheritance of DNA methylation patterns may play an important role in the stability of the differentiated state. We have therefore studied the inhibitory effects of DNA binding proteins on DNA methylation in vitro. Mouse L1210 cells grown in the presence of 5-azacytidine acquire hemimethylated sites in their DNA. Purified hemimethylated DNA accepted methyl groups from S-adenosyl-L-methionine in the presence of a crude maintenance methylase more readily than purified DNA isolated from cells not exposed to 5-azacytidine. On the other hand, chromatin fractions isolated from cells grown in the presence or absence of 5-azacytidine were poor substrates for the maintenance methylase irrespective of the number of hemimethylated sites present in the DNA. Inhibition of DNA methylation was shown to be associated primarily with chromatin proteins bound to DNA, and trypsinization of nuclei increased their methyl accepting abilities. Methyl acceptance was increased by salt extraction of chromosomal proteins. These data suggest that association of histones with DNA may play a role in the modulation of methylation patterns.     

A code controlling specific binding of regulatory proteins to DNA

A possible code is suggested that describes a correspondence between amino acid sequences in stereospecific sites of regulatory proteins and nucleotide sequences at the control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel -sheet with single-stranded regions at the ends of the -structure. The binding reaction between regulatory protein and double-helical DNA is accompanied by significant structural alterations at stereospecific sites of the protein and DNA. Half of the hydrogen bonds normally existing in -structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. The code states a correspondence between four amino acid residues at a stereospecific site of the regulatory protein and an AT (GC) base pair at the control site. It predicts that there are six fundamental amino acid residues (serine, threonine, histidine, asparagine, glutamine and cysteine) whose arrangement in the stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially.     

A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.     

PSSM-based prediction of DNA binding sites in proteins

Background  

Detection of DNA-binding sites in proteins is of enormous interest for technologies targeting gene regulation and manipulation. We have previously shown that a residue and its sequence neighbor information can be used to predict DNA-binding candidates in a protein sequence. This sequence-based prediction method is applicable even if no sequence homology with a previously known DNA-binding protein is observed. Here we implement a neural network based algorithm to utilize evolutionary information of amino acid sequences in terms of their position specific scoring matrices (PSSMs) for a better prediction of DNA-binding sites.     

Structure and function of poly(A) binding proteins

Poly (A) tails are found at the 3' ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5' end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.     

DNA binding proteins from Tetrahymena thermophila

We have used DNA-cellulose chromatography to isolate single-strand binding proteins from Tetrahymena thermophila. Three major proteins which bind to denatured DNA-cellulose were obtained. The predominant protein has a molecular weight of 20 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis and possesses many of the properties of the helix destabilizing proteins isolated from prokaryotic and eukaryotic sources. The protein facilitates denaturation of the synthetic copolymer poly[d(A-T).d(A-T)], depressing the melting temperature by nearly 40 degrees C. It also permits the renaturation of poly[d(A-T)].d(A-T)] in high salt concentration. Two other binding proteins have molecular weight of 25 000 and 23 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The protein with a molecular weight of 25 000 is probably the "M protein" previously isolated from Tetrahymena thermophila which has been shown to stimulate Tetrahymena DNA polymerase. These two proteins failed to show helix destabilizing, DNA dependent ATPase, or deoxyribonuclease activities. These three proteins are abundant in the cell with approximately 1.0 x 10(6) to 10.0 x 10(6) molecules of each protein monomer per cell. One molecule of each protein monomer binds to 7 to 10 nucleotides as detected by a nitrocellulose filter binding assay. Peptide mapping of the three proteins suggests that they are all distinct. We have also found that the binding proteins can interact with Tetrahymena DNA polymerase and some other proteins to form an enzyme complex, a putative replication complex.