Efficient expression and purification of human replication fork-stabilizing factor, Claspin, from mammalian cells: DNA-binding activity and novel protein interactions

Abstract

Purification of recombinant proteins of a large size often poses problems of instability or low expression in bacterial or insect cells. Here, we established a method for a high-level expression of large-sized recombinant proteins in mammalian cells and subsequent purification of the full-length proteins. We applied this method to express human Claspin and Tim-Tipin complex, which play important roles in replication checkpoint responses as fork-stabilizing factors, and successfully purified them in functional forms in amount sufficient for enzymatic characterization. Purified Claspin behaves as a monomer and binds preferentially to fork-like DNA. Over-expression of tagged Claspin in mammalian cells facilitated the detection of its interacting factors. Claspin interacts with many factors involved in checkpoint regulation and replication fork machinery, including ATR, ATM, Chk1, Tim, MCM4, MCM10, Cdc45, DNA polymerases a, d, e and Cdc7 kinase. We will discuss the potential implication of these findings in architecture of replication fork. We will also discuss the advantage of this system for purification and characterization of those proteins that are large and have been difficult to deal with.

And‐1 coordinates with Claspin for efficient Chk1 activation in response to replication stress

“…Claspin by itself displays little affinity for ssDNA (Sar et al, 2004;Kemp et al, 2010;Uno & Masai, 2011), and the stable association of Claspin with ssDNA is facilitated by an interaction between Tipin and RPA (Kemp et al, 2010). Our studies revealed that And-1 is another factor that regulates the association of Claspin with ssDNA.…”

“…Thus, we conclude that And-1 binds preferentially to ssDNA. Claspin alone displays little affinity for ssDNA and a Tipin-RPA interaction was shown to stabilize the association of Claspin with ssDNA, thereby promoting Claspin-mediated Chk1 activation (Sar et al, 2004;Kemp et al, 2010;Uno & Masai, 2011). Given that And-1 directly binds to ssDNA (Fig 6A) and that And-1 interacts with Timeless-Tipin and Claspin (Fig 1 and Supplementary Fig S1), we determined whether And-1 facilitates the association of Claspin with ssDNA.…”

“…Recent studies indicate that Tipin interacts with RPA and that this interaction stabilizes the association of Timeless-Tipin and Claspin with RPA-coated ssDNA to promote Claspin-mediated phosphorylation of Chk1 by ATR (Kemp et al, 2010). Interestingly, Claspin also physically interacts with Chk1 (Kumagai & Dunphy, 2000;Uno & Masai, 2011). Since Claspin interacts with both ATR and Chk1 (Kumagai & Dunphy, 2000;Chini & Chen, 2003), it is most likely that Claspin functions as an adaptor to bring Chk1 to ATR for Chk1 phosphorylation (Kumagai et al, 2004).…”

Dbf4 Is Direct Downstream Target of Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3-related (ATR) Protein to Regulate Intra-S-phase Checkpoint

“…It was also observed that DDK binds to Swi1/Tof1 and Swi3/Csm3, the components of the fork protection complex, and its kinase activity is required for induction and maintenance of the hyperphosphorylation of another fork protection protein, Mrc1, upon replication fork block (24,55,65). Similarly, in mammalian cells, recent studies showed that Cdc7 interacts with the fork-stabilizing factor Claspin and that Cdc7-mediated phosphorylation of Claspin is needed for ATR-mediated phosphorylation upon replication stress (66,67). Second, the kinase activity of DDK may be needed to attenuate checkpoint signaling and promote DNA repair.…”

The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity

“…The ORF sequence was confirmed by sequencing analysis (PRIMM; Milan, Italy). The plasmid expressing the truncated form of human Tim (FLAGTim5) was already described (28,29). The baculoviruses expressing the human DNA polymerase ⑀ subunits (p261, FLAG-p59, p17, and p12) were a generous gift from J. Hurwitz (Memorial Sloan Kettering Cancer Center, New York, NY).…”