ATRIP Oligomerization Is Required for ATR-dependent Checkpoint Signaling

Ball, Heather L.; Chen, David

doi:10.1074/jbc.m504961200

Cited by 75 publications

(93 citation statements)

References 32 publications

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“…Here, we show that xATR and xATM also control DNA damage-induced phosphorylation of xFANCM; moreover, xATR is required to recruit xFANCM to undamaged replicating chromatin. We were surprised to find that the absence of xATR in egg extracts also blocked chromatin recruitment of xFANCA, because we observed previously that depletion of xATRIP, the binding partner and "functional subunit" of xATR (22,24), does not inhibit chromatin binding of xFANCA (14) (or xFANCM). 4 However, although depletion of xATRIP inactivates the xATR kinase, it only partially co-depletes xATR from egg extracts.…”

Section: Discussionmentioning

confidence: 99%

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

Sobeck¹,

Stone

Landais

et al. 2009

Journal of Biological Chemistry

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In addition to monoubiquitination by the FA core complex, FANCD2 and FANCI are phosphorylated by the two major cell cycle checkpoint kinases, ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related),y in response to DNA damage (2-6). ATM-dependent phosphorylation of FANCD2 occurs following ionizing irradiation and is required for activation of the ionizing irradiation-induced intra-S phase checkpoint (4). ATR-dependent phosphorylation of FANCD2 is triggered by various types of DNA damage, including replication stress, and is required for the interstrand cross-link-induced intra-S phase checkpoint response (2, 3). Moreover, phosphorylation by ATR is required for efficient FANCD2 monoubiquitination in response to DNA damage, suggesting that the FA pathway might participate in ATR-dependent coordination of the S phase of the cell cycle (3, 7).The recent identification of a highly conserved FA core complex member, FANCM (8, 9), indicates a direct role of FA pathway proteins in repair steps at sites of DNA damage. FANCM is a homolog of the archaebacterial Hef protein (helicase-associated endonuclease for fork-structured DNA) and contains two DNA processing domains: a DEAH box helicase domain and an XPF/ERCC4-like endonuclease domain. FANCM has ATP-dependent DNA translocase activity and can dissociate DNA triple helices in vitro (8). Moreover, FANCM binds Holliday junctions and DNA replication fork structures in vitro and promotes ATP-dependent branch point migration, suggesting that FANCM might be involved in DNA processing at stalled replication forks (10,11). In human cells, FANCM localizes to chromatin and is required for chromatin recruitment of other FA core complex proteins (8,12). FANCM is phosphorylated during both the M and S phases and in response to DNA-damaging agents (8,12,13). Interestingly, DNA damage-induced phosphorylation of FANCM is independent of the FA core complex (8), suggesting that FANCM is controlled by other, as yet unknown upstream components of the DNA damage response. Here, we used cell-free Xenopus egg extracts to investigate the role of FANCM during replication and in the DNA damage response. We show that Xenopus FANCM (xFANCM) binds chromatin in a replication-dependent manner and is phosphorylated during unperturbed replication as well as in response to various DNA damage structures. Both chromatin recruitment and phosphorylation of xFANCM are partially controlled by xFANCD2, suggesting feedback signaling from xFANCD2 to the upstream xFA core complex via regulation of xFANCM. In addition, chromatin recruitment during unperturbed replication and activation of xFANCM in response to DNA damage are controlled by the xATR and xATM cell cycle kinases.

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Section: Discussionmentioning

confidence: 99%

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

Sobeck¹,

Stone

Landais

et al. 2009

Journal of Biological Chemistry

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“…PI3K-related kinases (PIKKs) such as ataxia telangiectasia mutated (ATM), ATM and Rad3-related protein (ATR), and DNA-dependent protein kinase (DNA-PK) are known to oligomerize (4)(5)(6). Biochemical and genetic analyses have identified self-association of mTOR and its orthologs in yeast and Drosophila (7)(8)(9)(10).…”

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confidence: 99%

Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown

Jain

Arauz²,

Aggarwal

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The mammalian target of rapamycin (mTOR) kinase is a master regulator of cellular, developmental, and metabolic processes. Deregulation of mTOR signaling is implicated in numerous human diseases including cancer and diabetes. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking. We use the single-molecule pulldown (SiMPull) assay that combines principles of conventional pulldown assays with single-molecule fluorescence microscopy to investigate the stoichiometry and assembly of mTORCs. After validating our approach with mTORC1, confirming a dimeric assembly as previously reported, we show that all major components of mTORC2 exist in two copies per complex, indicating that mTORC2 assembles as a homodimer. Interestingly, each mTORC component, when free from the complexes, is present as a monomer and no single subunit serves as the dimerizing component. Instead, our data suggest that dimerization of mTORCs is the result of multiple subunits forming a composite surface. SiMPull also allowed us to distinguish complex disassembly from stoichiometry changes. Physiological conditions that abrogate mTOR signaling such as nutrient deprivation or energy stress did not alter the stoichiometry of mTORCs. On the other hand, rapamycin treatment leads to transient appearance of monomeric mTORC1 before complete disruption of the mTOR-raptor interaction, whereas mTORC2 stoichiometry is unaffected. These insights into assembly of mTORCs may guide future mechanistic studies and exploration of therapeutic potential. mTOR | mTORC | single molecule | stoichiometry | rapamycin

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“…Using a combination of biochemical mapping, NMR, mutagenesis and computational modeling we have identified a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs. Mutations in the CRD of the yeast ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA damaging agents, and partially compromise checkpoint signaling (2). These data demonstrate that the CRD is critical for localization and optimal DNA damage responses.…”

Section: Bodymentioning

confidence: 73%

“…The recent finding by Kumagai et al that ATR can be activated by TopBP1 helped to fill in some of the missing pieces to the puzzle of how ATR is activated. TopBP1-dependent ATR activation is dependent on ATRIP but not on the RPA-binding domain of ATRIP, suggesting that TopBP1-dependent ATR activation is separate from RPA-dependent ATR-ATRIP recruitment (2,12). These findings lead to a revised, two-step model of ATR activation and have helped to change the premise in the field as to how ATR checkpoint signaling is activated (12).…”

Section: Discussionmentioning

confidence: 99%

Maintenance of Genome Stability and Breast Cancer: Molecular Analysis of DNA Damage-Activated Kinases

Mordes¹,

Ball²,

Ehrhardt³

et al. 2007

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBERVanderbilt University Medical Center Nashville, TN 37232 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTESOriginal contains colored plates: ALL DTIC reproductions will be in black and white. ABSTRACTThe ATR (ATM and Rad3-Related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (Replication Protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs has been identified by biochemical mapping of the RPA binding site in combination with NMR, mutagenesis and computational modeling. Mutations in the CRD of the yeast ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of TopBP1 to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support a multi-step model for ATR activation that requires separable localization and activation functions of ATRIP.

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ATRIP Oligomerization Is Required for ATR-dependent Checkpoint Signaling

Cited by 75 publications

References 32 publications

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown

Maintenance of Genome Stability and Breast Cancer: Molecular Analysis of DNA Damage-Activated Kinases

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