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1.
EMBO J ; 36(11): 1605-1622, 2017 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-28373211

RESUMEN

Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a major sub-pathway of conventional long-patch BER that involves formation of a 9-nucleotide gap 5' to the lesion. This new sub-pathway is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 poly(ADP-ribose) polymerase and RPA The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto-(ADP-ribosyl)ation and the choice between long-patch and single-nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long-patch BER and a new key regulation point for pathway choice in BER.


Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , RecQ Helicasas/metabolismo , Proteína de Replicación A/metabolismo , Línea Celular , ADN/metabolismo , Daño del ADN , Humanos , Modelos Biológicos
2.
Biochem J ; 468(2): 227-44, 2015 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-25774876

RESUMEN

RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in replication restart mechanisms. Cellular phenotypes of RECQ1 deficiency are indicative of aberrant repair of stalled replication forks, but the molecular functions of RECQ1, the most abundant of the five known human RecQ homologues, have remained poorly understood. We show that RECQ1 associates with FEN-1 (flap endonuclease-1) in nuclear extracts and exhibits direct protein interaction in vitro. Recombinant RECQ1 significantly stimulated FEN-1 endonucleolytic cleavage of 5'-flap DNA substrates containing non-telomeric or telomeric repeat sequence. RECQ1 and FEN-1 were constitutively present at telomeres and their binding to the telomeric chromatin was enhanced following DNA damage. Telomere residence of FEN-1 was dependent on RECQ1 since depletion of RECQ1 reduced FEN-1 binding to telomeres in unperturbed cycling cells. Our results confirm a conserved collaboration of human RecQ helicases with FEN-1 and suggest both overlapping and specialized roles of RECQ1 in the processing of DNA structure intermediates proposed to arise during replication, repair and recombination.


Asunto(s)
Cromatina/metabolismo , Endonucleasas de ADN Solapado/metabolismo , RecQ Helicasas/metabolismo , Telómero/metabolismo , Cromatina/genética , Inmunoprecipitación de Cromatina , Daño del ADN , Replicación del ADN , Endonucleasas de ADN Solapado/genética , Células HeLa , Humanos , Unión Proteica , RecQ Helicasas/genética , Telómero/genética
3.
Biochemistry ; 50(30): 6567-78, 2011 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-21714498

RESUMEN

Pin1 is a prolyl isomerase that recognizes phosphorylated Ser/Thr-Pro sites, and phosphatase inhibitor-2 (I-2) is phosphorylated during mitosis at a PSpTP site that is expected to be a Pin1 substrate. However, we previously discovered I-2, but not phospho-I-2, bound to Pin1 as an allosteric modifier of Pin1 substrate specificity [Li, M., et al. (2008) Biochemistry 47, 292]. Here, we use binding assays and NMR spectroscopy to map the interactions on Pin1 and I-2 to elucidate the organization of this complex. Despite having sequences that are ∼50% identical, human, Xenopus, and Drosophila I-2 proteins all exhibited identical, saturable binding to GST-Pin1 with K(0.5) values of 0.3 µM. The (1)H-(15)N heteronuclear single-quantum coherence spectra for both the WW domain and isomerase domain of Pin1 showed distinctive shifts upon addition of I-2. Conversely, as shown by NMR spectroscopy, specific regions of I-2 were affected by addition of Pin1. A single-residue I68A substitution in I-2 weakened binding to Pin1 by half and essentially eliminated binding to the isolated WW domain. On the other hand, truncation of I-2 to residue 152 had a minimal effect on binding to the WW domain but eliminated binding to the isomerase domain. Size exclusion chromatography revealed that wild-type I-2 and Pin1 formed a large (>300 kDa) complex and I-2(I68A) formed a complex of half the size that we propose are a heterotetramer and a heterodimer, respectively. Pin1 and I-2 are conserved among eukaryotes from yeast to humans, and we propose they make up an ancient partnership that provides a means for regulating Pin1 specificity and function.


Asunto(s)
Proteínas de Drosophila/química , Isomerasa de Peptidilprolil/química , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Proteínas de Xenopus/química , Regulación Alostérica/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Proteínas de Drosophila/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Unión Proteica , Multimerización de Proteína , Proteínas/química , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae , Especificidad por Sustrato/genética , Proteínas de Xenopus/metabolismo
4.
Mutat Res ; 790: 8-18, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27248010

RESUMEN

RecQ helicases are a highly conserved family of ATP-dependent DNA-unwinding enzymes with key roles in DNA replication and repair in all kingdoms of life. The RECQ1 gene encodes the most abundant RecQ homolog in humans. We engineered full-length RECQ1 harboring point mutations in the zinc-binding motif (amino acids 419-480) within the conserved RecQ-specific-C-terminal (RQC) domain known to be critical for diverse biochemical and cellular functions of RecQ helicases. Wild-type RECQ1 contains a zinc ion. Substitution of three of the four conserved cysteine residues that coordinate zinc severely impaired the ATPase and DNA unwinding activities but retained DNA binding and single strand DNA annealing activities. Furthermore, alteration of these residues attenuated zinc binding and significantly changed the overall conformation of full-length RECQ1 protein. In contrast, substitution of cysteine residue at position 471 resulted in a wild-type like RECQ1 protein. Differential contribution of the conserved cysteine residues to the structure and functions of the RECQ1 protein is also inferred by homology modeling. Overall, our results indicate that the zinc binding motif in the RQC domain of RECQ1 is a key structural element that is essential for the structure-functions of RECQ1. Given the recent association of RECQ1 mutations with breast cancer, these results will contribute to understanding the molecular basis of RECQ1 functions in cancer etiology.


Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Mutagénesis Sitio-Dirigida , Mutación Puntual , Dominios Proteicos/genética , RecQ Helicasas/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Secuencia Conservada , Proteínas de Unión al ADN/química , Escherichia coli/genética , Humanos , RecQ Helicasas/química , Alineación de Secuencia
5.
Comput Struct Biotechnol J ; 6: e201303014, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24688722

RESUMEN

The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in protecting the genome stability in all kingdoms of life. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5ß. Although the individual RecQ-related diseases are characterized by a variety of clinical features encompassing growth defects (Bloom Syndrome and Rothmund Thomson Syndrome) to premature aging (Werner Syndrome), all these patients have a high risk of cancer predisposition. Here, we present an overview of recent progress towards elucidating functions of RECQ1 helicase, the most abundant but poorly characterized RecQ homolog in humans. Consistent with a conserved role in genome stability maintenance, deficiency of RECQ1 results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased DNA damage and greater sensitivity to certain genotoxic stress. Delineating what aspects of RECQ1 catalytic functions contribute to the observed cellular phenotypes, and how this is regulated is critical to establish its biological functions in DNA metabolism. Recent studies have identified functional specialization of RECQ1 in DNA repair; however, identification of fundamental similarities will be just as critical in developing a unifying theme for RecQ actions, allowing the functions revealed from studying one homolog to be extrapolated and generalized to other RecQ homologs.

6.
FEBS Lett ; 587(21): 3406-11, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-24042052

RESUMEN

The deposition of fibrillated human islet ß-cell peptide islet amyloid polypeptide (hIAPP) into amyloid plaques is characteristic of the pathogenesis of islet cell death during type 2 diabetes. We investigated the effects of the neuroendocrine secretory proteins 7B2 and proSAAS on hIAPP fibrillation in vitro and on cytotoxicity. In vitro, 21-kDa 7B2 and proSAAS blocked hIAPP fibrillation. Structure-function studies showed that a central region within 21-kDa 7B2 is important in this effect and revealed the importance of the N-terminal region of proSAAS. Both chaperones blocked the cytotoxic effects of exogenous hIAPP on Rin5f cells; 7B2 generated by overexpression was also effective. ProSAAS and 7B2 may perform a chaperone role as secretory anti-aggregants in normal islet cell function and in type 2 diabetes.


Asunto(s)
Polipéptido Amiloide de los Islotes Pancreáticos/antagonistas & inhibidores , Polipéptido Amiloide de los Islotes Pancreáticos/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Animales , Células Cultivadas , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Ratones , Chaperonas Moleculares/metabolismo , Neuropéptidos , Ratas
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