Tipin-Replication Protein A Interaction Mediates Chk1 Phosphorylation by ATR in Response to Genotoxic Stress

Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.     

生物节律基因Timeless的生物学功能研究进展

Timeless基因广泛分布于生物体中,是主要的生物节律基因之一,它通过与节律基因Per和Cry家族成员的相互作用影响它们的表达水平。Timeless和Tipin能够稳定复制叉,促进姊妹染色单体凝聚,对DNA复制有促进作用;在细胞周期中激活S期检测点,参与ATR-Chk1和ATM-Chk2的DNA损伤修复通路,加强细胞周期的阻滞以修复DNA损伤。Timeless是生物节律和细胞周期的连接者,在多种癌组织(如肝癌、肺癌、乳腺癌、结直肠癌、肾癌和胰腺癌)中的表达水平与癌旁非癌组织相比有差异,提示Timeless表达异常可能与肿瘤的发生和发展相关。     

Molecular anatomy of the DNA damage and replication checkpoints

Cell cycle checkpoints are signal transduction pathways that enforce the orderly execution of the cell division cycle and arrest the cell cycle upon the occurrence of undesirable events, such as DNA damage, replication stress, and spindle disruption. The primary function of the cell cycle checkpoint is to ensure that the integrity of chromosomal DNA is maintained. DNA lesions and disrupted replication forks are thought to be recognized by the DNA damage checkpoint and replication checkpoint, respectively. Both checkpoints initiate protein kinase-based signal transduction cascade to activate downstream effectors that elicit cell cycle arrest, DNA repair, or apoptosis that is often dependent on dose and cell type. These actions prevent the conversion of aberrant DNA structures into inheritable mutations and minimize the survival of cells with unrepairable damage. Genetic components of the damage and replication checkpoints have been identified in yeast and humans, and a working model is beginning to emerge. We summarize recent advances in the DNA damage and replication checkpoints and discuss the essential functions of the proteins involved in the checkpoint responses.     

Human Tim/Timeless-interacting protein, Tipin, is required for efficient progression of S phase and DNA replication checkpoint

Tipin was originally isolated as a protein interacting with Timeless/Tim1/Tim (Tim), which is known to be involved in both circadian rhythm and cell cycle checkpoint regulation. The endogenous Tim and Tipin proteins in human cells, interacting through the N-terminal segment of each molecule, form a complex throughout the cell cycle. Tipin and Tim are expressed in the interphase nuclei mostly at constant levels during the cell cycle, and small fractions are recovered in the chromatin-enriched fractions during S phase. Depletion of endogenous Tipin results in reduced growth rate, and this may be due in part to inefficient progression of S phase and DNA synthesis. Knockdown of Tipin induces radioresistant DNA synthesis and inhibits phosphorylation of Chk1 kinase caused by replication stress, as was observed with that of Tim. Knockdown of Tipin or Tim results in reduced protein level and relocation to the cytoplasm of the respective binding partner, suggesting that the complex formation may be required for stabilization and nuclear accumulation of both proteins. Furthermore, both Tipin and Tim may facilitate the accumulation of Claspin in the nuclei under replication stress, whereas nuclear localization of Tipin and Tim is unaffected by Claspin. Our results indicate that mammalian Tipin is a checkpoint mediator that cooperates with Tim and may regulate the nuclear relocation of Claspin in response to replication checkpoint.     

A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.     

Novel Functions of the Phosphatase SHP2 in the DNA Replication and Damage Checkpoints

Replication stress- and DNA damage-induced cell cycle checkpoints are critical for maintaining genome stability. To identify protein phosphatases involved in the activation and maintenance of the checkpoints, we have carried out RNA interference-based screens with a human phosphatome shRNA library. Several phosphatases, including SHP2 (also called PTPN11) were found to be required for cell survival upon hydroxyurea-induced replicative stress in HeLa cells. More detailed studies revealed that SHP2 was also important for the maintenance of the checkpoint after DNA damage induced by cisplatin or ionizing radiation in HeLa cells. Furthermore, SHP2 was activated after replicative stress and DNA damage. Although depletion of SHP2 resulted in a delay in cyclin E accumulation and an extension of G₁ phase, these cell cycle impairments were not responsible for the increase in apoptosis after DNA damage. Depletion of SHP2 impaired CHK1 activation, checkpoint-mediated cell cycle arrest, and DNA repair. These effects could be rescued with a shRNA-resistant SHP2. These results underscore the importance of protein phosphatases in checkpoint control and revealed a novel link between SHP2 and cell cycle checkpoints.     

Modulation of ATR-mediated DNA damage checkpoint response by cryptochrome 1

Mammalian cryptochromes (Crys) are essential circadian clock factors implicated in diverse clock-independent physiological functions, including DNA damage responses. Here we show that Cry1 modulates the ATR-mediated DNA damage checkpoint (DDC) response by interacting with Timeless (Tim) in a time-of-day-dependent manner. The DDC capacity in response to UV irradiation showed a circadian rhythm. Interestingly, clock-deficient Cry1 and Cry2 double knockout (Cry^DKO) cells retained substantial DDC capacity compared with clock-proficient wild-type cells, although the Cry1-modulated oscillation of the DDC capacity was abolished in Cry^DKO cells. We found temporal interaction of Cry1 and Tim in the nucleus. When Cry1 was expressed in the nucleus, it was critical for circadian ATR activity. We regenerated rhythmic DDC responses by ectopically expressing Cry1 in Cry^DKO cells. In addition, we also investigated the DDC capacity in the liver of mice that were intraperitoneally injected with cisplatin at different circadian times (CT). When mice were injected at CT20, about 2-fold higher expression of phosphorylated minichromosome maintenance protein 2 (p-MCM2) was detected compared with mice injected at CT08, which consequently affected the removal rate of cisplatin-DNA adducts from genomic DNA. Taken together, our data demonstrate the intimate interaction between the circadian clock and the DDC system during genotoxic stress in clock-ticking cells.     

Chk1: a double agent in cell cycle checkpoints

Two cell cycle surveillance systems--the DNA damage checkpoint and the spindle checkpoint--guard against genomic instability. The protein kinase Chk1 is a well-established signal transducer in the DNA damage checkpoint. In this issue of Developmental Cell, Zachos et al.(2007) present evidence to indicate that Chk1 also plays a critical role in the spindle checkpoint, suggesting an interplay between the DNA damage and spindle checkpoints.     

Human TopBP1 ensures genome integrity during normal S phase

Cell cycle checkpoints are essential for maintaining genomic integrity. Human topoisomerase II binding protein 1 (TopBP1) shares sequence similarity with budding yeast Dpb11, fission yeast Rad4/Cut5, and Xenopus Cut5, all of which are required for DNA replication and cell cycle checkpoints. Indeed, we have shown that human TopBP1 participates in the activation of replication checkpoint and DNA damage checkpoints, following hydroxyurea treatment and ionizing radiation. In this study, we address the physiological function of TopBP1 in S phase by using small interfering RNA. In the absence of exogenous DNA damage, TopBP1 is recruited to replicating chromatin. However, TopBP1 does not appear to be essential for DNA replication. TopBP1-deficient cells have increased H2AX phosphorylation and ATM-Chk 2 activation, suggesting the accumulation of DNA double-strand breaks in the absence of TopBP1. This leads to formation of gaps and breaks at fragile sites, 4N accumulation, and aberrant cell division. We propose that the cellular function of TopBP1 is to monitor ongoing DNA replication. By ensuring proper DNA replication, TopBP1 plays a critical role in the maintenance of genomic stability during normal S phase as well as following genotoxic stress.     

细胞周期检控点

细胞周期是高度有组织的时序调控过程,受到DNA损伤检控点、DNA复制检控点和纺锤体检控点等细胞周期检控点的精确调控。细胞周期检控点的作用主要是调节细胞周期的时序转换,以确保DNA复制、染色体分离等细胞重要生命活动的高度精确性,并对DNA损伤、DNA复制受阻、纺锤体组装和染色体分离异常等细胞损伤及时做出反应,以防止突变和遗传不稳定的发生。细胞周期检控点的功能缺陷,将导致细胞基因组的不稳定,与细胞癌变密切相关。因此细胞周期检控点对于维持细胞遗传信息的稳定性和完整性以及防止细胞癌变和遗传疾病的发生起着至关重要的作用。     

Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation

Timeless was originally identified in Drosophila as an essential component of circadian cycle regulation, where its function is tightly controlled at the protein level by tyrosine phosphorylation and subsequent degradation. In mammals, Timeless has also been implicated in circadian rhythms as well as cell cycle control and embryonic development. Here we report that mammalian Timeless is an SH3 domain-binding protein and substrate for several members of the Src protein–tyrosine kinase family, including Fyn, Hck, c-Src and c-Yes. Co-expression of Tim with Fyn or Hck was followed by ubiquitylation and subsequent degradation in human 293 T cells. While c-Src and c-Yes also promoted Tim ubiquitylation, in this case ubiquitylation correlated with Tim protein accumulation rather than degradation. Both c-Src and c-Yes selectively promoted modification of Tim through Lys63-linked polyubiquitin, which may explain the differential effects on Tim protein turnover. These data show distinct and opposing roles for individual Src-family members in the regulation of Tim protein levels, suggesting a unique mechanism for the regulation of Tim function in mammals.     

Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway

DNA damage checkpoints lead to the inhibition of cell cycle progression following DNA damage. The Saccharomyces cerevisiae Mec1 checkpoint protein, a phosphatidylinositol kinase-related protein, is required for transient cell cycle arrest in response to DNA damage or DNA replication defects. We show that mec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Delta cells, indicating that the Mec1 conserved kinase domain is required for all known Mec1 functions, including cell viability and proper DNA damage response. Mec1kd variants maintain the ability to physically interact with both Ddc2 and wild-type Mec1 and cause dominant checkpoint defects when overproduced in MEC1 cells, impairing the ability of cells to slow down S phase entry and progression after DNA damage in G(1) or during S phase. Conversely, an excess of Mec1kd in MEC1 cells does not abrogate the G(2)/M checkpoint, suggesting that Mec1 functions required for response to aberrant DNA structures during specific cell cycle stages can be separable. In agreement with this hypothesis, we describe two new hypomorphic mec1 mutants that are completely defective in the G(1)/S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G(2). The finding that these mutants, although indistinguishable from mec1Delta cells with respect to the ability to replicate a damaged DNA template, do not lose viability after UV light and methyl methanesulfonate treatment suggests that checkpoint impairments do not necessarily result in hypersensitivity to DNA-damaging agents.     

Claspin, a regulator of Chk1 in DNA replication stress pathway

Regulation of the vertebrate checkpoint kinase Chk1 involves several protein complexes including the recently identified protein Claspin. Claspin associates with Chk1 upon replication stress and DNA damage and is required for Chk1 activation in both Xenopus and human systems. More importantly, Claspin is involved in regulation of cell cycle checkpoints. Here, we discuss the emerging roles of Claspin in the Chk1 pathway and its functions in checkpoint control.     

Assaying the DNA damage checkpoint in fission yeast

Cell cycle checkpoints exist to ensure the proper maintenance and stable inheritance of genomic information. The pathways that insure the faithful execution of these checkpoints are well conserved throughout evolution. In the fission yeast, Schizosaccharomyces pombe, a major cell cycle checkpoint exists that responds to the presence of damaged DNA and prevents this damage from being propagated to future generations. Fission yeast is an ideal system to investigate these pathways because there exist specific techniques that allow one to assay the fidelity of this DNA damage checkpoint pathway.     

“Isogaba Maware”: quality control of genome DNA by checkpoints

Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event. The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication. Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete. A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human. They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators. The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome. In humans, defects in the checkpoints are often associated with cancer-prone diseases. Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress. BioEssays 20 :391–399, 1998.© 1998 John Wiley & Sons Inc.     

The Human G2 Checkpoint Control Protein hRAD9 Is a Nuclear Phosphoprotein That Forms Complexes with hRAD1 and hHUS1

Eukaryotic cells actively block entry into mitosis in the presence of DNA damage or incompletely replicated DNA. This response is mediated by signal transduction cascades called cell cycle checkpoints. We show here that the human checkpoint control protein hRAD9 physically associates with two other checkpoint control proteins, hRAD1 and hHUS1. Furthermore, hRAD1 and hHUS1 themselves interact, analogously to their fission yeast homologues Rad1 and Hus1. We also show that hRAD9 is present in multiple phosphorylation forms in vivo. These phosphorylated forms are present in tissue culture cells that have not been exposed to exogenous sources of DNA damage, but it remains possible that endogenous damage or naturally occurring replication intermediates cause the observed phosphorylation. Finally, we show that hRAD9 is a nuclear protein, indicating that in this signal transduction pathway, hRAD9 is physically proximal to the upstream (DNA damage) signal rather than to the downstream, cytoplasmic, cell cycle machinery.