RESUMEN
The RecQL4 helicase is involved in the maintenance of genome integrity and DNA replication. Mutations in the human RecQL4 gene cause the Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. Mouse models and experiments in human and Xenopus have proven the N-terminal part of RecQL4 to be vital for cell growth. We have identified the first 54 amino acids of RecQL4 (RecQL4_N54) as the minimum interaction region with human TopBP1. The solution structure of RecQL4_N54 was determined by heteronuclear liquid-state nuclear magnetic resonance (NMR) spectroscopy (PDB 2KMU; backbone root-mean-square deviation 0.73 Å). Despite low-sequence homology, the well-defined structure carries an overall helical fold similar to homeodomain DNA-binding proteins but lacks their archetypical, minor groove-binding N-terminal extension. Sequence comparison indicates that this N-terminal homeodomain-like fold is a common hallmark of metazoan RecQL4 and yeast Sld2 DNA replication initiation factors. RecQL4_N54 binds DNA without noticeable sequence specificity yet with apparent preference for branched over double-stranded (ds) or single-stranded (ss) DNA. NMR chemical shift perturbation observed upon titration with Y-shaped, ssDNA and dsDNA shows a major contribution of helix α3 to DNA binding, and additional arginine side chain interactions for the ss and Y-shaped DNA.
Asunto(s)
ADN/metabolismo , Proteínas de Homeodominio/química , RecQ Helicasas/química , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , RecQ Helicasas/metabolismo , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Cartilla de ADN/genética , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Factores de Transcripción de Octámeros/química , Factores de Transcripción de Octámeros/genética , Factor de Empalme Asociado a PTB , Dominios y Motivos de Interacción de Proteínas , Proteómica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Topoisomerase IIbeta binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy-terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP-ribose) polymerase 1 (PARP-1), where the latter interaction is responsible for the poly(ADP-ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 A. The crystal structure reveals a degenerate phospho-peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho-peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho-peptide binding domain. We further provide insight into poly(ADP-ribose) binding and sites of potential modification by PARP-1.