RESUMO
PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain.
Assuntos
Motivos de Aminoácidos , DNA Helicases/química , Domínios Proteicos , Proteínas Repressoras/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , DNA Helicases/genética , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitose/genética , Modelos Moleculares , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Chromosome integrity depends on DNA structure-specific processing complexes that resolve DNA entanglement between sister chromatids. If left unresolved, these entanglements can generate either chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis. These bridge structures are defined by the presence of the PICH protein, which interacts with the BEND3 protein in mitosis. To obtain structural insights into PICH-BEND3 complex formation at the atomic level, their respective NTPR and BD1 domains were cloned, overexpressed and crystallized using 1.56â M ammonium sulfate as a precipitant at pH 7.0. The protein complex readily formed large hexagonal crystals belonging to space group P6122, with unit-cell parameters a = b = 47.28, c = 431.58â Å and with one heterodimer in the asymmetric unit. A complete multiwavelength anomalous dispersion (MAD) data set extending to 2.2â Å resolution was collected from a selenomethionine-labelled crystal at the Swiss Light Source.
Assuntos
DNA Helicases/química , Proteínas Repressoras/química , Selenometionina/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Códon/química , Códon/metabolismo , Cristalografia por Raios X , DNA Helicases/genética , DNA Helicases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Selenometionina/metabolismoRESUMO
DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing.
