Claspin – checkpoint adaptor and DNA replication factor

Abstract

Claspin was discovered as a Chk1-interacting protein necessary for Chk1 phosphorylation and activation by the upstream kinase, ATR, in response to DNA synthesis inhibition in Xenopus oocyte extracts. Subsequent investigations have defined a molecular model in which Claspin acts as an adaptor or scaffold protein to facilitate activation of Chk1 by ATR within a multiprotein complex that forms on single-stranded DNA at stalled replication forks and sites of DNA damage. Interestingly, Claspin is an unstable protein whose degradation via the proteasome is tightly regulated via ubiquitination and controlled by multiple ubiquitin ligases and deubiquitinases. As a result, Claspin levels fluctuate during the cell cycle, contributing to the regulation of checkpoint proficiency and playing a key role in terminating checkpoint-mediated cell cycle arrest. In addition to its role in signalling genotoxic stress, Claspin is required to maintain normal rates of replication fork progression during unperturbed DNA replication and may contribute to the regulation of replication origin firing. Consistent with this, Claspin can bind directly to DNA, with particular affinity for branched or forked molecules, and it interacts with multiple protein components of the replisome. As expected for a protein with key roles in checkpoint signalling and genome duplication, aberrations of Claspin expression and structure have been observed in cancer. Claspin is furthermore targeted to facilitate viral replication and plays a role in suppressing cellular DNA synthesis in response to nongenotoxic endoplasmic reticulum stress. Here, we review the functions and regulation of Claspin with a focus on areas of active research.

OZF is a Claspin‐interacting protein essential to maintain the replication fork progression rate under replication stress

“…Cell fractionation showed that OZF is a chromatin‐bound protein (Figure 3A). To investigate if this chromatin‐bound OZF interacts, similarly to Claspin, with replisome proteins, 18 immunoprecipitation of chromatin‐bound OZF was performed. As shown in Figure 3B, OZF from nuclear extract co‐immunoprecipitated not only with Claspin (Figure 1), but also with Cdc45, MCM6, and MCM2, components of the CMG complex.…”

“…We first identified OZF as a Claspin‐binding protein by a two‐hybrid analysis and the interaction was also confirmed by co‐immunoprecipitation with both exogenously expressed proteins and endogenous proteins. Claspin is a protein that interacts with several replisome components and travels with this protein complex in active replication forks while DNA is synthesized 18,22 . Thus, to examine whether OZF was also present in these structures, the interaction of OZF with replisome components was analyzed.…”

“…Claspin (and its yeast ortholog, Mrc1) is one of the proteins present in the replisome but it does not have a direct role in nucleotide incorporation 16‐18 . The protein complex formed by Claspin‐Tipin‐Timeless, called fork protection complex, represents a physical and functional link between CMG and polymerases, ensuring the stability of the replisome and coordinating polymerases and helicases, 19‐23 although the exact mechanism of action remains to be elucidated.…”

DNA damage kinase signaling: checkpoint and repair at 30 years

“…While it is currently unknown whether mammalian ATR directly phosphorylates Claspin, Xenopus ATR has been shown to directly phosphorylate Claspin at threonines 817 and 819, which are critical for CHK1 recruitment (Fig 2; the uncertainty of ATR's direct phosphorylation of Claspin in humans is denoted by "? "; Yoo et al, 2006; in-depth discussion of the similarities and differences between Claspin orthologs can be found here: Smits et al, 2019). Once bound to CHK1, Claspin is thought to both stabilize CHK1 and tether it in proximity to ATR, allowing for extensive phosphorylation and full activation of the effector kinase (Liu et al, 2006).…”

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells

“…Bertolli et al, have shown that oncogenic stimulation causes replication stress, and oppositely, sustained E2F-dependent transcription allows the cells to decrease replicative stress and remove the block for cell proliferation [63]. During oncogenic transformation, cells could utilize claspin, because it helps to circumvent replicative stress [64]. However, in this study, we have shown that an increase in c-Myc and possibly E2F transcription factors are decisive factors in aiding enhanced cell proliferation due to IHH stimulus.…”