Background: Although Cdc45 is a key replication factor, there are no biochemical or structural studies on the isolated protein.
Results:We report the first purification and biochemical characterization of human Cdc45, as well as the first structural data on the isolated Cdc45 by small angle x-ray scattering. Conclusion: Cdc45 is related to the RecJ/DHH family of phosphoesterases and binds single-stranded DNA. Significance: The similarity has important evolutionary implications.
GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM. This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble. Reconstruction of the double-tetrameric form, combined with small-angle X-ray scattering data, allows the localisation of the B domain of the Psf1 subunit in the free GINS complex, which was not visible in previous studies and is essential for the formation of a functional replication fork.
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