scholarly journals Roles of Replication Fork-interacting and Chk1-activating Domains from Claspin in a DNA Replication Checkpoint Response

2005 ◽  
Vol 16 (11) ◽  
pp. 5269-5282 ◽  
Author(s):  
Joon Lee ◽  
Daniel A. Gold ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
William G. Dunphy
Keyword(s):  
Replication Fork ◽  
Protein A ◽  
Replication Forks ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Dna Replication Checkpoint ◽  
Egg Extracts ◽  
High Concentrations ◽  
Factor C ◽  
Xenopus Egg Extracts

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. Claspin associates with replication forks upon origin unwinding. We show that Claspin contains a replication fork-interacting domain (RFID, residues 265–605) that associates with Cdc45, DNA polymerase ϵ, replication protein A, and two replication factor C complexes on chromatin. The RFID contains two basic patches (BP1 and BP2) at amino acids 265–331 and 470–600, respectively. Deletion of either BP1 or BP2 compromises optimal binding of Claspin to chromatin. Absence of BP1 has no effect on the ability of Claspin to mediate activation of Chk1. By contrast, removal of BP2 causes a large reduction in the Chk1-activating potency of Claspin. We also find that Claspin contains a small Chk1-activating domain (residues 776–905) that does not bind stably to chromatin, but it is fully effective at high concentrations for mediating activation of Chk1. These results indicate that stable retention of Claspin on chromatin is not necessary for activation of Chk1. Instead, our findings suggest that only transient interaction of Claspin with replication forks potentiates its Chk1-activating function. Another implication of this work is that stable binding of Claspin to chromatin may play a role in other functions besides the activation of Chk1.

scholarly journals The Human Tim/Tipin Complex Coordinates an Intra-S Checkpoint Response to UV That Slows Replication Fork Displacement

2007 ◽  
Vol 27 (8) ◽  
pp. 3131-3142 ◽  
Author(s):  
Keziban Ünsal-Kaçmaz ◽  
Paul D. Chastain ◽  
Ping-Ping Qu ◽  
Parviz Minoo ◽  
Marila Cordeiro-Stone ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Protein A ◽  
Chain Elongation ◽  
Replication Forks ◽  
Interacting Protein ◽  
Checkpoint Response ◽  
Replication Fork Progression ◽  
Dna Chain ◽  
Interfering Rna

ABSTRACT UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

The DNA replication checkpoint response stabilizes stalled replication forks

Nature ◽  
2001 ◽  
Vol 412 (6846) ◽  
pp. 557-561 ◽  
Author(s):  
Massimo Lopes ◽  
Cecilia Cotta-Ramusino ◽  
Achille Pellicioli ◽  
Giordano Liberi ◽  
Paolo Plevani ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Forks ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Dna Replication Checkpoint ◽  
Stalled Replication Forks

scholarly journals Replication Checkpoint Kinase Cds1 Regulates Recombinational Repair Protein Rad60

2003 ◽  
Vol 23 (16) ◽  
pp. 5939-5946 ◽  
Author(s):  
Michael N. Boddy ◽  
Paul Shanahan ◽  
W. Hayes McDonald ◽  
Antonia Lopez-Girona ◽  
Eishi Noguchi ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Integrity ◽  
Recombinational Repair ◽  
Replication Forks ◽  
Checkpoint Kinase ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Repair Protein ◽  
Replication Fork Arrest ◽  
Structural Maintenance Of Chromosomes

ABSTRACT Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks.

scholarly journals The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci

2000 ◽  
Vol 14 (1) ◽  
pp. 81-96 ◽  
Author(s):  
Christian Frei ◽  
Susan M. Gasser
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Replication Fork ◽  
Dna Helicase ◽  
S Phase ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Replication Fork Progression ◽  
Cycle Arrest ◽  
Dna Replication Checkpoint

We have examined the cellular function of Sgs1p, a nonessential yeast DNA helicase, homologs of which are implicated in two highly debilitating hereditary human diseases (Werner's and Bloom's syndromes). We show that Sgs1p is an integral component of the S-phase checkpoint response in yeast, which arrests cells due to DNA damage or blocked fork progression during DNA replication. DNA polε and Sgs1p are found in the same epistasis group and act upstream of Rad53p to signal cell cycle arrest when DNA replication is perturbed. Sgs1p is tightly regulated through the cell cycle, accumulates in S phase and colocalizes with Rad53p in S-phase-specific foci, even in the absence of fork arrest. The association of Rad53p with a chromatin subfraction is Sgs1p dependent, suggesting an important role for the helicase in the signal-transducing pathway that monitors replication fork progression.

scholarly journals Positive Regulation of Wee1 by Chk1 and 14-3-3 Proteins

2001 ◽  
Vol 12 (3) ◽  
pp. 551-563 ◽  
Author(s):  
Joon Lee ◽  
Akiko Kumagai ◽  
William G. Dunphy
Keyword(s):  
Cell Cycle ◽  
Active Form ◽  
Wild Type ◽  
Common Pathway ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
M Phase ◽  
Dna Replication Checkpoint ◽  
Egg Extracts ◽  
Positive Regulators

Wee1 inactivates the Cdc2–cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted fromXenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.

scholarly journals Fork Pausing Complex Engages Topoisomerases at the Replisome

2019 ◽  
Author(s):  
Maksym Shyian ◽  
Benjamin Albert ◽  
Andreja Moset Zupan ◽  
Vitalii Ivanitsa ◽  
Gabriel Charbonnet ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Topoisomerase Ii ◽  
Replication Forks ◽  
Replication Checkpoint ◽  
Slowing Down ◽  
C Terminus ◽  
Dna Replication Checkpoint ◽  
Molecular Brake

ABSTRACTReplication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) acts as a ‘molecular brake’ and promotes fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C-terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a ‘sTOP’ mechanism (‘slowing down with TOPoisomerases I-II’), which we show also contributes to protecting cells from topoisomerase-blocking agents.

scholarly journals Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling

2006 ◽  
Vol 173 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Shan Yan ◽  
Howard D. Lindsay ◽  
W. Matthew Michael
Keyword(s):  
Sperm Chromatin ◽  
Checkpoint Control ◽  
Direct Role ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Oligonucleotide Duplex ◽  
Stalled Replication Forks

TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3–related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.

scholarly journals Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

2020 ◽  
Vol 6 (38) ◽  
pp. eabc0330 ◽  
Author(s):  
D. T. Gruszka ◽  
S. Xie ◽  
H. Kimura ◽  
H. Yardimci
Keyword(s):  
Dna Replication ◽  
Real Time ◽  
Single Molecule ◽  
Replication Fork ◽  
Epigenetic Inheritance ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Fork Stalling ◽  
The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

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