scholarly journals The Mre11-Rad50-Nbs1 Complex Mediates Activation of TopBP1 by ATM

2009 ◽  
Vol 20 (9) ◽  
pp. 2351-2360 ◽  
Author(s):  
Hae Yong Yoo ◽  
Akiko Kumagai ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
William G. Dunphy
Keyword(s):  
Human Cells ◽  
Dna Breaks ◽  
Checkpoint Response ◽  
Mrn Complex ◽  
Functional Studies ◽  
Double Stranded Dna ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The activation of ATR-ATRIP in response to double-stranded DNA breaks (DSBs) depends upon ATM in human cells and Xenopus egg extracts. One important aspect of this dependency involves regulation of TopBP1 by ATM. In Xenopus egg extracts, ATM associates with TopBP1 and thereupon phosphorylates it on S1131. This phosphorylation enhances the capacity of TopBP1 to activate the ATR-ATRIP complex. We show that TopBP1 also interacts with the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. This interaction involves the Nbs1 subunit of the complex. ATM can no longer interact with TopBP1 in Nbs1-depleted egg extracts, which suggests that the MRN complex helps to bridge ATM and TopBP1 together. The association between TopBP1 and Nbs1 involves the first pair of BRCT repeats in TopBP1. In addition, the two tandem BRCT repeats of Nbs1 are required for this binding. Functional studies with mutated forms of TopBP1 and Nbs1 suggested that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. These findings suggest that the MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs.

scholarly journals CtIP interacts with TopBP1 and Nbs1 in the response to double-stranded DNA breaks (DSBs) in Xenopus egg extracts

Cell Cycle ◽  
2011 ◽  
Vol 10 (3) ◽  
pp. 469-480 ◽  
Author(s):  
Juan S. Ramírez-Lugo ◽  
Hae Yong Yoo ◽  
Su Jin Yoon ◽  
William G. Dunphy
Keyword(s):  
Dna Breaks ◽  
Double Stranded Dna ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases

2004 ◽  
Vol 165 (6) ◽  
pp. 801-812 ◽  
Author(s):  
Wenhui Li ◽  
Soo-Mi Kim ◽  
Joon Lee ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Chromosomal Abnormalities ◽  
Sister Chromatid Exchanges ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Chromatid Exchanges ◽  
Chromosomal Defects

Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIα (Xtop3α) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3α has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.

scholarly journals ZW10 links mitotic checkpoint signaling to the structural kinetochore

2005 ◽  
Vol 169 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Geert J.P.L. Kops ◽  
Yumi Kim ◽  
Beth A.A. Weaver ◽  
Yinghui Mao ◽  
Ian McLeod ◽  
...  
Keyword(s):  
Chromosome Instability ◽  
Mitotic Checkpoint ◽  
Human Cells ◽  
Structural Elements ◽  
Checkpoint Signaling ◽  
Daughter Cells ◽  
Primary Mechanism ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The mitotic checkpoint ensures that chromosomes are divided equally between daughter cells and is a primary mechanism preventing the chromosome instability often seen in aneuploid human tumors. ZW10 and Rod play an essential role in this checkpoint. We show that in mitotic human cells ZW10 resides in a complex with Rod and Zwilch, whereas another ZW10 partner, Zwint-1, is part of a separate complex of structural kinetochore components including Mis12 and Ndc80–Hec1. Zwint-1 is critical for recruiting ZW10 to unattached kinetochores. Depletion from human cells or Xenopus egg extracts is used to demonstrate that the ZW10 complex is essential for stable binding of a Mad1–Mad2 complex to unattached kinetochores. Thus, ZW10 functions as a linker between the core structural elements of the outer kinetochore and components that catalyze generation of the mitotic checkpoint-derived “stop anaphase” inhibitor.

scholarly journals Xenopus HJURP and condensin II are required for CENP-A assembly

2011 ◽  
Vol 192 (4) ◽  
pp. 569-582 ◽  
Author(s):  
Rafael Bernad ◽  
Patricia Sánchez ◽  
Teresa Rivera ◽  
Miriam Rodríguez-Corsino ◽  
Ekaterina Boyarchuk ◽  
...  
Keyword(s):  
Protein A ◽  
Human Cells ◽  
Epigenetic Mark ◽  
Temporal Specificity ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Centromeric Protein ◽  
Xenopus Egg Extracts

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction–recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.

scholarly journals Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts

Cell Cycle ◽  
2012 ◽  
Vol 11 (6) ◽  
pp. 1183-1194 ◽  
Author(s):  
Sanjay Kumar ◽  
Hae Yong Yoo ◽  
Akiko Kumagai ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
...  
Keyword(s):  
Checkpoint Response ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Damaged Dna

scholarly journals DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

2002 ◽  
Vol 158 (5) ◽  
pp. 863-872 ◽  
Author(s):  
Matthew P. Stokes ◽  
Ruth Van Hatten ◽  
Howard D. Lindsay ◽  
W. Matthew Michael
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Alkylating Agents ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Damaged Dna

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.

scholarly journals MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication

2017 ◽  
Vol 28 (22) ◽  
pp. 2998-3012 ◽  
Author(s):  
Akiko Kumagai ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
G4 Dna ◽  
G Quadruplex ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication ◽  
Xenopus Egg Extracts

Treslin, which is essential for incorporation of Cdc45 into the replicative helicase, possesses a partner called MTBP (Mdm2-binding protein). We have analyzed Xenopus and human MTBP to assess its role in DNA replication. Depletion of MTBP from Xenopus egg extracts, which also removes Treslin, abolishes DNA replication. These extracts be can rescued with recombinant Treslin-MTBP but not Treslin or MTBP alone. Thus, Treslin-MTBP is collectively necessary for replication. We have identified a C-terminal region of MTBP (the CTM domain) that binds efficiently to both double-stranded DNA and G-quadruplex (G4) DNA. This domain also exhibits homology with budding yeast Sld7. Mutants of MTBP without a functional CTM domain are defective for DNA replication in Xenopus egg extracts. These mutants display an impaired localization to chromatin and the inability to support loading of Cdc45. Human cells harboring such a mutant also display severe S-phase defects. Thus, the CTM domain of MTBP plays a critical role in localizing Treslin-MTBP to the replication apparatus for initiation.

scholarly journals Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopus egg extracts

2007 ◽  
Vol 1773 (4) ◽  
pp. 556-564 ◽  
Author(s):  
Yuji Kumata ◽  
Shusuke Tada ◽  
Yumie Yamanada ◽  
Takashi Tsuyama ◽  
Takayuki Kobayashi ◽  
...  
Keyword(s):  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Double Strand Dna

scholarly journals The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks

2013 ◽  
Vol 24 (9) ◽  
pp. 1343-1353 ◽  
Author(s):  
Joon Lee ◽  
William G. Dunphy
Keyword(s):  
Substantial Reduction ◽  
Nuclease Activity ◽  
Replication Forks ◽  
Wild Type ◽  
Mrn Complex ◽  
Egg Extracts ◽  
Unknown Property ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Stalled Replication Forks

The activation of Chk1 in response to stalled replication forks in Xenopus egg extracts involves a complex pathway containing ATM and Rad3-related (ATR), topoisomerase IIβ-binding protein 1 (TopBP1), Rad17, the Rad9-Hus1-Rad1 (9-1-1) complex, and Claspin. We have observed that egg extracts lacking the Mre11-Rad50-Nbs1 (MRN) complex show greatly, although not completely, reduced activation of Chk1 in response to replication blockages. Depletion of both Rad17 and MRN leads to a further, essentially complete, reduction in the activation of Chk1. Thus, Rad17 and MRN act in at least a partially additive manner in promoting activation of Chk1. There was not an obvious change in the binding of RPA, ATR, Rad17, or the 9-1-1 complex to chromatin in aphidicolin (APH)-treated, MRN-depleted extracts. However, there was a substantial reduction in the binding of TopBP1. In structure–function studies of the MRN complex, we found that the Mre11 subunit is necessary for the APH-induced activation of Chk1. Moreover, a nuclease-deficient mutant of Mre11 cannot substitute for wild-type Mre11 in this process. These results indicate that the MRN complex, in particular the nuclease activity of Mre11, plays an important role in the activation of Chk1 in response to stalled replication forks. These studies reveal a previously unknown property of the MRN complex in genomic stability.

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