title: In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair.
abstract: Chromatin is the substrate for many processes in the cell nucleus, including transcription, replication, and various DNA repair systems, all of which require the formation of multiprotein machineries on the chromatin fiber. We have analyzed the kinetics of in vivo assembly of the protein complex that is responsible for nucleotide excision repair (NER) in mammalian cells. Assembly is initiated by UV irradiation of a small area of the cell nucleus, after which the accumulation of GFP-tagged NER proteins in the DNA-damaged area is measured, reflecting the establishment of the dual-incision complex. The dynamic behavior of two NER proteins, ERCC1-XPF and TFIIH, was studied in detail. Results show that the repair complex is assembled with a rate of approximately 30 complexes per second and is not diffusion limited. Furthermore, we provide in vivo evidence that not only binding of TFIIH, but also its helicase activity, is required for the recruitment of ERCC1-XPF. These studies give quantitative insight into the de novo assembly of a chromatin-associated protein complex in living cells.
authors.affiliation: Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.
authors.forename: Martijn J
authors.initials: MJ
authors.lastname: Moné
authors.forename: Tytus
authors.initials: T
authors.lastname: Bernas
authors.forename: Christoffel
authors.initials: C
authors.lastname: Dinant
authors.forename: Feliks A
authors.initials: FA
authors.lastname: Goedvree
authors.forename: Erik M M
authors.initials: EM
authors.lastname: Manders
authors.forename: Marcel
authors.initials: M
authors.lastname: Volker
authors.forename: Adriaan B
authors.initials: AB
authors.lastname: Houtsmuller
authors.forename: Jan H J
authors.initials: JH
authors.lastname: Hoeijmakers
authors.forename: Wim
authors.initials: W
authors.lastname: Vermeulen
authors.forename: Roel
authors.initials: R
authors.lastname: van Driel
mesh_terms: D000818:Animals; D016466:CHO Cells; D002843:Chromatin / Q000378:metabolism* / Q000528:radiation effects; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051758:Transcription Factor TFIIH; D019473:Transcription Factors, TFII / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019473:Transcription Factors, TFII; D049452:Green Fluorescent Proteins; D051758:Transcription Factor TFIIH; D004720:Endonucleases