Abstract
Human Timeless is involved in replication fork stabilization, S-phase checkpoint activation and establishment of sister chromatid cohesion. In the cell, Timeless forms a constitutive heterodimeric complex with Tipin. Here we present the 1.85 Å crystal structure of a large N-terminal segment of human Timeless, spanning amino acids 1–463, and we show that this region of human Timeless harbours a partial binding site for Tipin. Furthermore, we identify minimal regions of the two proteins that are required for the formation of a stable Timeless–Tipin complex and provide evidence that the Timeless–Tipin interaction is based on a composite binding interface comprising different domains of Timeless.
Highlights
DNA replication in eukaryotes is performed by a large and dynamic protein assembly known as the replisome [1]
As no homologous structure was available in the protein data bank (PDB) that could be used as template for molecular replacement, the structure was determined using the anomalous signal in diffraction data of crystals soaked with K2PtCl4
The findings reported here provide a first, partial highresolution description of Timeless and a biochemical analysis of its interaction with Tipin, which will be necessary to understand the important role of this complex in maintaining genomic stability during DNA replication
Summary
DNA replication in eukaryotes is performed by a large and dynamic protein assembly known as the replisome [1]. The normal progress of DNA replication can be challenged by alterations in the DNA template, such as DNA damage and unusual secondary structures, or the presence of protein–DNA barriers, which can lead to replication fork stalling and possible collapse, eventually threatening genomic integrity [2] To cope with such eventuality, the replisome is equipped with non-enzymatic components that are not directly involved in DNA synthesis, but are thought to be essential for maintaining replisome integrity and modulating replisome function during replicative stress [3]. Timeless displays an evolutionary-conserved interaction with another replisome component, Tipin [8,9]; Tipin and its yeast orthologue Csm have been ascribed roles in DNA synthesis that mirror those of Tof1 [7,10,11,12] Following these initial observation, experimental evidence has established that the metazoan Timeless–Tipin complex has important roles in genotoxic stress resistance and checkpoint activation [13,14,15]
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