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1.
Open Biol ; 11(6): 210060, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34129781

RESUMO

The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.


Assuntos
Endodesoxirribonucleases/química , Conformação Proteica , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Mutação , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes , Análise Espectral , Relação Estrutura-Atividade , Difração de Raios X
2.
Biochemistry ; 33(6): 1300-10, 1994 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-8312247

RESUMO

The capacity of the globular domain of the chicken erythrocyte linker histone H5 (GH5) to self-associate in solution has been demonstrated by chemical cross-linking with dimethyl 3,3'-dithiobis-(propionimidate) (DTBP), dithiobis(succinimidyl propionate) (DSP), and 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP). Several observations suggest that the GH5-GH5 interactions that mediate self-association are specific: (a) Incubation with each of the above reagents produces a discrete and characteristic pattern of cross-linked products; (b) GH1, the related peptide from chicken erythrocyte H1, is not cross-linked under the same conditions; (c) GH5 is not cross-linked with disuccinimidyl tartrate (DST), which has a shorter cross-linking span (6.4 A) than the other reagents (12 A); and (d) analysis of cross-linking as a function of peptide concentration provides an equilibrium constant for GH5 self-association of (4.8 +/- 1.3) x 10(3) M-1. The ability of GH5 to specifically self-associate is compatible with the proposal [Thoma, F., Koller, T., & Klug, A. (1979) J. Cell Biol. 83, 403-427] that linker histone globular domains occupy an axial position within the higher order chromatin fiber; the spatial juxtaposition of the GH5 domains at this location would be expected to promote their association and exert a stabilizing effect upon higher order chromatin structure.


Assuntos
Histonas/química , Animais , Galinhas/sangue , Dicroísmo Circular , Reagentes de Ligações Cruzadas , Eritrócitos/química , Concentração de Íons de Hidrogênio , Imidoésteres , Substâncias Macromoleculares , Modelos Moleculares , Concentração Osmolar , Dobramento de Proteína , Estrutura Secundária de Proteína , Cloreto de Sódio/farmacologia , Soluções , Succinimidas , Tripsina/farmacologia
3.
J Mol Biol ; 223(3): 651-9, 1992 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-1542112

RESUMO

The globular domain of the linker histone H5 has been expressed in Escherichia coli. The purified peptide is functional as it permits chromatosome protection during micrococcal nuclease digestion of chromatin reconstituted with the peptide, indicating that it binds correctly at the dyad axis of the nucleosomal core particle. The globular domain residue lysine 64 is highly conserved within the linker histone family, and site-directed mutagenesis has been used to assess the importance of this residue in the binding of the globular domain of linker histone H5 to the nucleosome. Recombinant peptides mutated at lysine 64 are unable to elicit chromatosome protection to the same degree as the wild-type peptide, and since they appear to be fully folded, these observations confirm a major role for this residue in determining the effective interaction between the globular domain of histone H5 and the nucleosome.


Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Galinhas , Cromatina/química , Clonagem Molecular , DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Histonas/química , Histonas/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tripsina
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