title: Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex.
abstract: The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork.
authors.affiliation: Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Justine
authors.initials: J
authors.lastname: Witosch
authors.affiliation: Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Department of Physiological Chemistry and Center For Integrated Protein Science Munich (CIPSM), Butenandt Institute, Ludwig Maximilians University of Munich, Butenandtstrasse 5, 81377 Munich, Germany Institut für allgemeine Botanik, Johannes Gutenberg-University, Johannes-von-Müller-Weg 6, 55128 Mainz, Germany and Institute of Molecular Biology (IMB), Mainz, Germany mizuno@biochem.mpg.de.
authors.forename: Eva
authors.initials: E
authors.lastname: Wolf
authors.affiliation: Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany mizuno@biochem.mpg.de.
authors.forename: Naoko
authors.initials: N
authors.lastname: Mizuno
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D051716:Replication Protein A; C509770:Timeless protein, mouse; C518120:Tipin protein, mouse