Human Claspin Is a Ring-shaped DNA-binding Protein with High Affinity to Branched DNA Structures

Abstract

Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.

Cleavage of Claspin by Caspase-7 during Apoptosis Inhibits the Chk1 Pathway

“…Claspin has a predicted molecular mass of 151 kDa, but migrates anomalously on SDS-polyacrylamide gel with an apparent molecular mass of ϳ250 kDa as noted previously (26). In a human cell-free system made from a mixture of nuclear and cytoplasmic extracts of HeLa cells (29) in which caspases are activated by the addition of cytochrome c (35), Claspin was modified from the apparent 250-kDa form to a doublet with an apparent molecular mass of ϳ220 kDa, suggesting that it was being proteolytically cleaved (Fig.…”

“…A number of other proteins involved in responses to DNA damage, including PARP (52), ATM (38), and DNA-dependent protein kinase (53)(54)(55)(56), are also inactivated by caspase cleavage during apoptosis by separation of functional domains. Previous work has identified a putative DNA-binding motif in the N-terminal half of Claspin that is conserved in yeast Mrc1 (26,27), although another, weaker, direct interaction with DNA through the C-terminal region was also found (26). Our results indicate that a region contained within the C-terminal 260 amino acids of human Claspin is essential for interaction with DNA in a cell-free system in which the checkpoint pathway to Chk1 is functional.…”

“…Claspin interacts with chromatin during S phase, indicating that it may also act as a sensor of DNA replication (24,25). Although Claspin has a DNAbinding motif in its N-terminal half (26), similar to Mrc1 (27), it is not clear if this direct interaction with DNA occurs in cells. The association of Claspin with chromatin during S phase involves the pre-replication complex and Cdc45 (24).…”

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

“…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.…”

“…Like TimelessTipin, Claspin is also important for efficient DNA replication in unperturbed cells (Lee et al, 2003;Sar et al, 2004;Petermann et al, 2008). 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).…”