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Involvement of p54(nrb), a PSF partner protein,in DNA double-strand break repair and radioresistance
Shuyi Li Wendy W. Kuhne Anita Kulharya Farlyn Z. Hudson Kyungsoo Ha Zhen Cao William S. Dynan 《Nucleic acids research》2009,37(20):6746-6753
Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a γ-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development. 相似文献
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The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF.p54(nrb) complex cooperates with Ku protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF.p54(nrb) complex cooperate to align separate DNA molecules. 相似文献
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PSF and p54(nrb)/NonO--multi-functional nuclear proteins 总被引:13,自引:0,他引:13
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Kuhnert A Schmidt U Monajembashi S Franke C Schlott B Grosse F Greulich KO Saluz HP Hänel F 《Journal of cellular biochemistry》2012,113(5):1744-1753
TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by co-immunoprecipitation assays and LC-ESI-MS-analyses. As TopBP1 binding partners we identified p54(nrb) and PSF, and confirmed the physical interactions by GST pull-down assays, co-immunoprecipitations and by yeast two-hybrid experiments. Recent evidence shows an involvement of p54(nrb) and PSF in DNA double-strand break repair (DSB) and radioresistance. To get a first picture of the physiological significance of the interaction of TopBP1 with p54(nrb) and PSF we investigated in real time the spatiotemporal behaviour of the three proteins after laser microirradiation of living cells. Localisation of TopBP1 at damage sites was noticed as early as 5 s following damage induction, whereas p54(nrb) and PSF localised there after 20 s. Both p54(nrb) and PSF disappeared after 20 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair. 相似文献
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PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP. 相似文献
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Tuncer Arslan Anil T. Abraham Sidney M. Hecht 《Nucleosides, nucleotides & nucleic acids》2013,32(1-3):515-530
Abstract A partial DNA duplex containing a high efficiency topoisomerase I cleavage site was substituted singly at each of three sites with 3′-deoxyadenosine. Depending on the site of substitution, the facility of the topoisomerase I-mediated cleavage or ligation reactions was altered. Inclusion of the modified nucleoside at the 5′-end of the acceptor oligonucleotide diminished the rate of religation following substrate cleavage by the enzyme. 相似文献
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Sequences in PSF/SFPQ mediate radioresistance and recruitment of PSF/SFPQ-containing complexes to DNA damage sites in human cells 总被引:1,自引:0,他引:1
PSF (gene name SFPQ) is a member of a small family of proteins with dual functions in RNA biogenesis and DNA repair. PSF and PSF-containing complexes stimulate double-strand break repair in cell free systems, most likely via direct interaction with the repair substrate. Prior in vitro studies are, however, insufficient to demonstrate whether PSF contributes to DNA repair in living cells. Here, we investigate the effect of miRNA-mediated PSF knockdown in human (HeLa) cells. We find that PSF is essential for reproductive viability. To circumvent this and investigate the DNA damage sensitivity phenotype, we established a genetic rescue assay based on co-transfection of PSF miRNA and mutant PSF expression constructs. Mutational analysis suggests that sequences required for viability and radioresistance are partially separable, and that the latter requires a unique N-terminal PSF domain. As an independent means to investigate PSF sequences involved in DNA repair, we established an assay based on real-time relocalization of PSF-containing complexes to sites of dense, laser-induced DNA damage in living cells. We show that relocalization is driven by sequences in PSF, rather than its dimerization partner, p54(nrb)/NONO, and that sequences required for relocalization reside in the same N-terminal domain that contributes to radioresistance. Further evidence for the importance of PSF sequences in mediating relocalization is provided by observations that PSF promotes relocalization of a third protein, PSPC1, under conditions where p54(nrb) is limiting. Together, these observations support the model derived from prior biochemical studies that PSF influences repair via direct, local, interaction with the DNA substrate. 相似文献
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Bromberg KD Hendricks C Burgin AB Osheroff N 《The Journal of biological chemistry》2002,277(34):31201-31206
Despite the importance of topoisomerase II-mediated DNA ligation to the essential physiological functions of the enzyme, the mechanistic details of this important reaction are poorly understood. Because topoisomerase II normally does not release cleaved DNA molecules prior to ligation, it is not known whether all of the nucleic acid specificity of its cleavage/ligation cycle is embodied in DNA cleavage or whether ligation also contributes specificity to the enzyme. All currently available ligation assays require that topoisomerase II cleave the initial DNA substrate before rejoining can be monitored. Consequently, it has been impossible to examine the specificity of DNA ligation separately from that of scission. To address this issue, a cleavage-independent topoisomerase II DNA ligation assay was developed. This assay utilizes a nicked oligonucleotide whose 5'-phosphate terminus at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. Human topoisomerase IIalpha and enzymes with active-site mutations that abrogated cleavage activity ligated the activated nick by catalyzing the direct attack of the terminal 3'-OH on the activated 5'-phosphate. Results with different DNA sequences indicate that human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for ligation that parallels its specificity for DNA cleavage. 相似文献
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To identify new potential substrates for the MAP kinase signal-integrating kinases (Mnks), we employed a proteomic approach. The Mnks are targeted to the translational machinery through their interaction with the cap-binding initiation factor complex. We tested whether proteins retained on cap resin were substrates for the Mnks in vitro, and identified one such protein as PSF (the PTB (polypyrimidine tract-binding protein)-associated splicing factor). Mnks phosphorylate PSF at two sites in vitro, and our data show that PSF is an Mnk substrate in vivo. We also demonstrate that PSF, together with its partner, p54(nrb), binds RNAs that contain AU-rich elements (AREs), such as those for proinflammatory cytokines (e.g. tumor necrosis factor alpha (TNFalpha)). Indeed, PSF associates specifically with the TNFalpha mRNA in living cells. PSF is phosphorylated at two sites by the Mnks. Our data show that Mnk-mediated phosphorylation increases the binding of PSF to the TNFalpha mRNA in living cells. These findings identify a novel Mnk substrate. They also suggest that the Mnk-catalyzed phosphorylation of PSF may regulate the fate of specific mRNAs by modulating their binding to PSF.p54(nrb). 相似文献
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Purification and cDNA cloning of HeLa cell p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6. 总被引:5,自引:0,他引:5
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While searching for a human homolog of the S.cerevisiae splicing factor PRP18, we found a polypeptide that reacted strongly with antibodies against PRP18. We purified this polypeptide from HeLa cells using a Western blot assay, and named it p54nrb (for nuclear RNA-binding protein, 54 kDa). cDNAs encoding p54nrb were cloned with probes derived from partial sequence of the purified protein. These cDNAs have identical coding sequences but differ as a result of alternative splicing in the 5' untranslated region. The cDNAs encode a 471 aa polypeptide that contains two RNA recognition motifs (RRMs). Human p54nrb has no homology to yeast PRP18, except for a common epitope, but is instead 71% identical to human splicing factor PSF within a 320 aa region that includes both RRMs. In addition, both p54nrb and PSF are rich in Pro and Gln residues outside the main homology region. The Drosophila puff-specific protein BJ6, one of three products encoded by the alternatively spliced no-on-transient A gene (nonA), which is required for normal vision and courtship song, is 42% identical to p54nrb in the same 320 aa region. The striking homology between p54nrb, PSF, and NONA/BJ6 defines a novel phylogenetically conserved protein segment, termed DBHS domain (for Drosophila behavior, human splicing), which may be involved in regulating diverse pathways at the level of pre-mRNA splicing. 相似文献
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J Q Svejstrup K Christiansen I I Gromova A H Andersen O Westergaard 《Journal of molecular biology》1991,222(3):669-678
A new technique for uncoupling the cleavage and religation half-reactions of topoisomerase I at a specific site has been developed. The technique takes advantage of a suicidal DNA substrate to attain enzyme-mediated cleavage without concomitant religation. Efficient religation can be achieved, subsequently, by addition of an oligonucleotide capable of hybridising to the non-cleaved strand of the suicide DNA substrate. The technique was used to study the effect of different compounds on the half-reactions of topoisomerase I. It was shown that topoisomerase I-mediated cleavage was inhibited by NaCl concentrations higher than 200 mM, while the religation reaction seemed unaffected by concentrations as high as 3 M-NaCl. The divalent cations Mg2+, Ca2+ and Mn2+ were found to enhance the cleavage but not the religation reaction of topoisomerase I, whereas Cu2+ and Zn2+ inhibited both reactions. Furthermore, the effect of the anti-neoplastic agent, camptothecin, on the half-reactions of topoisomerase I was investigated. It was found that the drug did not affect the cleavage reaction of topoisomerase I at the studied site, while the religation reaction of the enzyme was inhibited. Camptothecin was found to stabilise the enzyme-DNA cleavage complex even when the drug was added after complex formation. 相似文献
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Ito T Watanabe H Yamamichi N Kondo S Tando T Haraguchi T Mizutani T Sakurai K Fujita S Izumi T Isobe T Iba H 《The Biochemical journal》2008,411(1):201-209
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The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion. 相似文献