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Lene Juel Rasmussen - One of the best experts on this subject based on the ideXlab platform.
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Exonuclease 1 and its versatile roles in dna repair
Critical Reviews in Biochemistry and Molecular Biology, 2016Co-Authors: Guido Keijzers, Dekang Liu, Lene Juel RasmussenAbstract:Exonuclease 1 (EXO1) is a multifunctional 5' → 3' Exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions and biological roles of EXO1, its possible protective role against cancer and aging, and regulation of EXO1 by posttranslational modification.
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human Exonuclease 1 exo1 activity characterization and its function on flap structures
Bioscience Reports, 2015Co-Authors: Guido Keijzers, Vilhelm A Bohr, Lene Juel RasmussenAbstract:Human Exonuclease 1 (EXO1) is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of EXO1 have been described in yeast. Here, we report a biochemical characterization of human full-length EXO1. Prior to assay EXO1 on different DNA flap structures, we determined factors essential for the thermodynamic stability of EXO1. We show that enzymatic activity and stability of EXO1 on DNA is modulated by temperature. By characterization of EXO1 flap activity using various DNA flap substrates, we show that EXO1 has a strong capacity for degrading double stranded DNA and has a modest endonuclease or 5′ flap activity. Furthermore, we report novel mechanistic insights into the processing of flap structures, showing that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of a forked and nicked DNA flap substrates, suggesting a possible role of EXO1 in strand displacement.
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bi directional routing of dna mismatch repair protein human Exonuclease 1 to replication foci and dna double strand breaks
DNA Repair, 2011Co-Authors: Sascha Emilie Liberti, Guido Keijzers, Finn Cilius Nielsen, Vilhelm A Bohr, Sofie Dabros Andersen, Jing Wang, Simona Miron, Mylene Perderiset, Jeanbaptiste Charbonnier, Lene Juel RasmussenAbstract:Abstract Human Exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ( 788 QIKLNELW 795 ). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390–490 and 787–846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.
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nuclear localization of human dna mismatch repair protein Exonuclease 1 hexo1
Nucleic Acids Research, 2007Co-Authors: Nina Ostergaard Knudsen, Finn Cilius Nielsen, Lena Vinther, Ronni Bertelsen, Steen Holtenandersen, Sascha Emilie Liberti, Robert M W Hofstra, Krista Kooi, Lene Juel RasmussenAbstract:Human Exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence 418KRPR421, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin beta/alpha1,3,7 whereas hMSH2 specifically recognizes importin beta/alpha3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.
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characterization of human Exonuclease 1 in complex with mismatch repair proteins subcellular localization and association with pcna
Oncogene, 2004Co-Authors: Finn Cilius Nielsen, Anne Charlotte Jager, Anne Lutzen, Jens R Bundgaard, Lene Juel RasmussenAbstract:Human Exonuclease 1 (hEXO1) has been implicated in DNA mismatch repair (MMR), replication, and recombination, but the nature of its interaction with these cellular processes is still ambiguous. We show that hEXO1 colocalizes with proliferating cell nuclear antigen (PCNA) at DNA replication sites and that the C-terminal region of hEXO1 is sufficient for this localization. We also show that both hMLH1-hPMS2 (MutLalpha) and hMLH1-hEXO1 complexes are formed in a reaction mixture containing all three proteins. Moreover, hEXO1 5' double-stranded Exonuclease activity on a homoduplex substrate but not on a substrate containing a G/T mismatch was inhibited by complex formation with hMSH2-hMSH6 (MutSalpha) or MutLalpha. Taken together, the results support a model in which hEXO1 plays a role in events at the replication sites as well as a functional role in the MMR and/or recombination processes.
Binghui Shen - One of the best experts on this subject based on the ideXlab platform.
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functional alterations of human Exonuclease 1 mutants identified in atypical hereditary nonpolyposis colorectal cancer syndrome
Cancer Research, 2002Co-Authors: Xuemin Sun, Li Zheng, Binghui ShenAbstract:Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is a genetically heterogeneous disorder caused by germ-line mutations in one of several DNA mismatch repair (MMR) genes, most commonly in hMSH2 and hMLH1 . Human Exonuclease 1 (hExo1) possesses both 5′Exonuclease and flap endonuclease activities and plays a role in DNA repair, recombination, and replication. The enzyme interacts with MMR proteins, hMsh2, hMlh1, and hMsh3. Recently, eight missense mutations in hEXO1 were identified in atypical HNPCC patients, who have been screened to be negative for hMSH2 , hMLH1 , and hMSH6 mutations. To address the question of whether these mutations cause susceptibility to HNPCC, in vitro nuclease activity and protein-protein interaction assays were performed in this study. We found that two mutants, E109K and L410R, lost their Exonuclease activities while retaining their capacity to bind to the DNA substrate. Three other mutants, P640S, G759E, and P770L, displayed a reduced capacity to interact with hMsh2. The combination of these three point mutations leads to the binding capacity with hMsh2 to nearly zero. Evidence made available in this study sheds light on the pathogenesis of HNPCC, perhaps initiated by an additional MMR gene, hEXO1 .
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human Exonuclease 1 functionally complements its yeast homologues in dna recombination rna primer removal and mutation avoidance
Journal of Biological Chemistry, 1999Co-Authors: Junzhuan Qiu, Ying Qian, Victoria Chen, Minxin Guan, Binghui ShenAbstract:Yeast Exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways. The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1. In this report, we show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays. Furthermore, the human enzyme suppresses the conditional lethality of a rad27Delta mutant, symptomatic of defective RNA primer removal. The purified recombinant enzyme not only displays 5'-3' double strand DNA Exonuclease activity, but also shows an RNase H activity. This result indicates a back-up function of Exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.
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human Exonuclease 1 functionally complements its yeast homologues in dna recombination rna primer removal and mutation avoidance
Journal of Biological Chemistry, 1999Co-Authors: Junzhuan Qiu, Ying Qian, Victoria Chen, Minxin Guan, Binghui ShenAbstract:Yeast Exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways. The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1. In this report, we show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays. Furthermore, the human enzyme suppresses the conditional lethality of a rad27Δ mutant, symptomatic of defective RNA primer removal. The purified recombinant enzyme not only displays 5′-3′ double strand DNA Exonuclease activity, but also shows an RNase H activity. This result indicates a back-up function of Exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.
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saccharomyces cerevisiae Exonuclease 1 plays a role in uv resistance that is distinct from nucleotide excision repair
Nucleic Acids Research, 1998Co-Authors: Junzhuan Qiu, Minxin Guan, Adam M Bailis, Binghui ShenAbstract:Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of the RAD2 gene in S. cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains. In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease. Expression of the EXO1 gene is UV inducible. Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants. Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA. The exo1 msh2 , exo1 rad51 , rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants. This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER. Further, it suggests that the EXO1 , RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.
Stefano Ferrari - One of the best experts on this subject based on the ideXlab platform.
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the human Exonuclease 1 interactome and phosphorylation sites
Biochemical and Biophysical Research Communications, 2019Co-Authors: Wassim Eid, Christiane Konig, Daniel Hess, Christian Gentili, Stefano FerrariAbstract:Error-free repair of DNA double-strand breaks is orchestrated by homologous recombination (HR) pathways and requires the concerted action of several factors. Among these, Exonulcease-1 (EXO1) and DNA2/BLM execute extensive resection of DNA ends to produce 3'-overhangs, which are key intermediates for downstream steps of HR. To help shedding light on regulatory aspects of DNA repair pathways in which EXO1 participates, we set out to identify proteins interacting with EXO1. Affinity purification of EXO1 followed by Orbitrap mass spectrometry led to the identification of novel partners that are involved in RNA processing or that are the causative agents of rare X-linked disorders. Depletion of a selected subset of EXO1 interacting proteins led to reduction of the DNA damage response. Among those, we examined the RRP5-homologue and NF-kappa-B-interacting protein PDCD11/ALG-4, which has roles in apoptosis and is a putative driver gene in cutaneous T-cell lymphoma. We provide evidence that depletion of PDCD11 decreased the formation of γ-H2AX foci and the phosphorylation of DNA damage response (DDR) signaling intermediates in response to camptothecin or bleomycin, resulting in increased cellular resistance to DNA damage. Furthermore, extensive coverage of EXO1 sequence by mass spectrometry allowed conducting an in-depth analysis of its phosphorylation sites, with the identification of 26 residues that are differentially modified in untreated conditions or upon induction of DNA damage.
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the human Exonuclease 1 interactome and phosphorylation sites
bioRxiv, 2019Co-Authors: Wassim Eid, Christiane Konig, Daniel Hess, Christian Gentili, Stefano FerrariAbstract:Error-free repair of DNA double-strand break is orchestrated by homologous recombination (HR) pathways and requires the concerted action of several factors. Among these, EXO1 and DNA2/BLM execute extensive resection of DNA ends to produce 3-overhangs, which are key intermediates for downstream steps of HR. To help shedding light on regulatory aspects of DNA repair pathways in which EXO1 participates, we set out to identify proteins interacting with EXO1. Affinity purification of EXO1 followed by Orbitrap mass spectrometry led to the identification of novel partners that are involved in RNA processing or that are the causative agents of rare X-linked disorders. Depletion of a selected subset of EXO1 interacting proteins led to reduction of the DNA damage response. Among those, we examined the RRP5-homologue and NF{kappa}B-interacting protein PDCD11/ALG-4, which has roles in apoptosis and is a putative driver gene in cutaneous T-cell lymphoma. We provide evidence that depletion of PDCD11 decreased the formation of {gamma}H2AX foci and the phosphorylation of DNA damage response signaling intermediates in response to camptothecin or bleomycin, resulting in increased cellular resistance to DNA damage. Furthermore, extensive coverage of EXO1 sequence (>85%) by mass spectrometry allowed conducting an in-depth analysis of its phosphorylation sites, with the identification of 26 residues that are differentially modified in untreated conditions or upon induction of DNA damage.nnAs a whole, these results provide the basis for future in-depth studies on novel roles of EXO1 in genome stability and indicate targets for pharmacological inhibition of pathways of cancer development.nnHIGHLIGHTS O_LIProteome-wide analysis of Exonuclease-1 (EXO1) interacting proteins revealed novel partners involved in RNA processing or that are the causative agents of rare X-linked disorders.nC_LIO_LIWe provide evidence for a role of PDCD11 in the DNA Damage Response.nC_LIO_LIWe conducted a comprehensive identification of EXO1 phosphorylation sites.nC_LI
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14 3 3 checkpoint regulatory proteins interact specifically with dna repair protein human Exonuclease 1 hexo1 via a semi conserved motif
DNA Repair, 2012Co-Authors: Sofie Dabros Andersen, Guido Keijzers, Mahmoud Elshemerly, Finn Cilius Nielsen, Vilhelm A Bohr, Kim Engels, Emmanouil Rampakakis, Patricia Luhn, Alfred May, Stefano FerrariAbstract:Human Exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.
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14 3 3 proteins regulate Exonuclease 1 dependent processing of stalled replication forks
PLOS Genetics, 2011Co-Authors: Kim Engels, Michele Giannattasio, Marco Muzifalconi, Massimo Lopes, Stefano FerrariAbstract:Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1) processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3–deficient cells fail to induce Mec1–dependent Exo1 hyperphosphorylation and accumulate Exo1–dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress.
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dna end resection by ctip and Exonuclease 1 prevents genomic instability
EMBO Reports, 2010Co-Authors: Wassim Eid, Martin Steger, Mahmoud Elshemerly, Lorenza P Ferretti, Javier Penadiaz, Christiane Konig, Emanuele Valtorta, Alessandro A Sartori, Stefano FerrariAbstract:End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and Exonucleases such as Exonuclease 1 (EXO1). In this study, we show that the localization of EXO1 to DSBs depends on both CtIP and MRN. We also establish that CtIP interacts with EXO1 and restrains its exonucleolytic activity in vitro. Finally, we show that on exposure to camptothecin, depletion of EXO1 in CtIP-deficient cells increases the frequency of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity.
Jane A Grasby - One of the best experts on this subject based on the ideXlab platform.
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human Exonuclease 1 threads 5 flap substrates through its helical arch
Biochemistry, 2017Co-Authors: Steven J Shaw, David L Finger, Jane A GrasbyAbstract:Human Exonuclease 1 (hEXO1) is a member of the 5′-nuclease superfamily and plays important roles in DNA repair. Along with acting as a 5′-Exonuclease on blunt, gapped, nicked, and 3′-overhang DNAs, hEXO1 can also act as an endonuclease removing protruding 5′-single-stranded flaps from duplex ends. How hEXO1 and related 5′-nuclease human flap endonuclease 1 (hFEN1) are specific for discontinuous DNA substrates like 5′-flaps has been controversial. Here we report the first functional data that imply that hEXO1 threads the 5′-flap through a hole in the protein known as the helical arch, thereby excluding reactions of continuous single strands. Conjugation of bulky 5′-streptavidin that would “block” threading through the arch drastically slowed the hEXO1 reaction. In contrast, addition of streptavidin to a preformed hEXO1 5′-biotin flap DNA complex trapped a portion of the substrate in a highly reactive threaded conformation. However, another fraction behaves as if it were “blocked” and decayed very slowly, i...
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Human Exonuclease 1 Threads 5′-Flap Substrates through Its Helical Arch
2017Co-Authors: Steven J. Shaw, David L Finger, Jane A GrasbyAbstract:Human Exonuclease 1 (hEXO1) is a member of the 5′-nuclease superfamily and plays important roles in DNA repair. Along with acting as a 5′-Exonuclease on blunt, gapped, nicked, and 3′-overhang DNAs, hEXO1 can also act as an endonuclease removing protruding 5′-single-stranded flaps from duplex ends. How hEXO1 and related 5′-nuclease human flap endonuclease 1 (hFEN1) are specific for discontinuous DNA substrates like 5′-flaps has been controversial. Here we report the first functional data that imply that hEXO1 threads the 5′-flap through a hole in the protein known as the helical arch, thereby excluding reactions of continuous single strands. Conjugation of bulky 5′-streptavidin that would “block” threading through the arch drastically slowed the hEXO1 reaction. In contrast, addition of streptavidin to a preformed hEXO1 5′-biotin flap DNA complex trapped a portion of the substrate in a highly reactive threaded conformation. However, another fraction behaves as if it were “blocked” and decayed very slowly, implying there were both threaded and unthreaded forms of the substrate present. The reaction of an unmodified hEXO1–flap DNA complex did not exhibit marked biphasic kinetics, suggesting a fast re-equilibration occurs that produces more threaded substrate when some decays. The finding that a threading mechanism like that used by hFEN1 is also used by hEXO1 unifies the mode of operation for members of the 5′-nuclease superfamily that act on discontinuous substrates. As with hFEN1, intrinsic disorder of the arch region of the protein may explain how flaps can be threaded without a need for a coupled energy source
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unpairing and gating sequence independent substrate recognition by fen superfamily nucleases
Trends in Biochemical Sciences, 2012Co-Authors: Jane A Grasby, John A Tainer, David L Finger, Susan E Tsutakawa, John M AtackAbstract:Structure-specific 5′-nucleases form a superfamily of evolutionarily conserved phosphodiesterases that catalyse a precise incision of a diverse range of DNA and RNA substrates in a sequence-independent manner. Superfamily members, such as flap endonucleases, Exonuclease 1, DNA repair protein XPG, endonuclease GEN1 and the 5′-3′-exoribonucleases, play key roles in many cellular processes such as DNA replication and repair, recombination, transcription, RNA turnover and RNA interference. In this review, we discuss recent results that highlight the conserved architectures and active sites of the structure-specific 5′-nucleases. Despite substrate diversity, a common gating mechanism for sequence-independent substrate recognition and incision emerges, whereby double nucleotide unpairing of substrates is required to access the active site.
Finn Cilius Nielsen - One of the best experts on this subject based on the ideXlab platform.
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14 3 3 checkpoint regulatory proteins interact specifically with dna repair protein human Exonuclease 1 hexo1 via a semi conserved motif
DNA Repair, 2012Co-Authors: Sofie Dabros Andersen, Guido Keijzers, Mahmoud Elshemerly, Finn Cilius Nielsen, Vilhelm A Bohr, Kim Engels, Emmanouil Rampakakis, Patricia Luhn, Alfred May, Stefano FerrariAbstract:Human Exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.
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bi directional routing of dna mismatch repair protein human Exonuclease 1 to replication foci and dna double strand breaks
DNA Repair, 2011Co-Authors: Sascha Emilie Liberti, Guido Keijzers, Finn Cilius Nielsen, Vilhelm A Bohr, Sofie Dabros Andersen, Jing Wang, Simona Miron, Mylene Perderiset, Jeanbaptiste Charbonnier, Lene Juel RasmussenAbstract:Abstract Human Exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ( 788 QIKLNELW 795 ). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390–490 and 787–846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.
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nuclear localization of human dna mismatch repair protein Exonuclease 1 hexo1
Nucleic Acids Research, 2007Co-Authors: Nina Ostergaard Knudsen, Finn Cilius Nielsen, Lena Vinther, Ronni Bertelsen, Steen Holtenandersen, Sascha Emilie Liberti, Robert M W Hofstra, Krista Kooi, Lene Juel RasmussenAbstract:Human Exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence 418KRPR421, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin beta/alpha1,3,7 whereas hMSH2 specifically recognizes importin beta/alpha3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.
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characterization of human Exonuclease 1 in complex with mismatch repair proteins subcellular localization and association with pcna
Oncogene, 2004Co-Authors: Finn Cilius Nielsen, Anne Charlotte Jager, Anne Lutzen, Jens R Bundgaard, Lene Juel RasmussenAbstract:Human Exonuclease 1 (hEXO1) has been implicated in DNA mismatch repair (MMR), replication, and recombination, but the nature of its interaction with these cellular processes is still ambiguous. We show that hEXO1 colocalizes with proliferating cell nuclear antigen (PCNA) at DNA replication sites and that the C-terminal region of hEXO1 is sufficient for this localization. We also show that both hMLH1-hPMS2 (MutLalpha) and hMLH1-hEXO1 complexes are formed in a reaction mixture containing all three proteins. Moreover, hEXO1 5' double-stranded Exonuclease activity on a homoduplex substrate but not on a substrate containing a G/T mismatch was inhibited by complex formation with hMSH2-hMSH6 (MutSalpha) or MutLalpha. Taken together, the results support a model in which hEXO1 plays a role in events at the replication sites as well as a functional role in the MMR and/or recombination processes.