Protein-protein interactions, sub-cellular localisation and mobility of the human Cdc45 protein.

Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays a role in the intra-S-phase checkpoint. Its interactions with a variety of replication proteins forming two central complexes, Cdc45/Mcm2-7/GINS (CMG) the putative replicative helicase of eukaryotes, and replisome progression complex (RPC) during the cell cycle and after intra-S-phase checkpoint activation remain to be fully characterized. The C terminal part of Cdc45 is important for its interaction with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A (RPA) and DNA polymerase ¿ are maximal during S phase. Following UVC-mediated DNA damage, Cdc45-Claspin complex formation is reduced whereas the binding of Cdc45 to RPA is not affected. We also show that treatment of cells with UCN-01, Caffeine or Wortmannin does not rescue the UV-mediated destabilisation of Cdc45-Claspin interactions, suggesting that the loss of interaction between Cdc45 and Claspin occurs independently of ATR activation in the intra-S-phase checkpoint. The sub-cellular localisation of Cdc45 in the cell cycle and following activation of the intra-S-phase checkpoint was also determined. Cdc45 showed a specific nuclear and nucleolar localisation and the Cdc45 association with nucleoli was abolished following UV damage and inhibiting nucleolar transcription. We then determined the regions of Cdc45 needed for its localisation to nuclear compartments. Spcifically, aa101-155 and aa156-169 are required for recruitment of Cdc45 to the nucleus and nucleolus, respectively. Measuring the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase showed that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. These findings suggest that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage

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