ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

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Sensitivity of tumor cells deficient in the fanconi anemia pathway to inhibition of ataxia telangiectasia mutated (ATM)

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.10509 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 10509-10509

Author(s):

R. D. Kennedy ◽

P. Stuckert ◽

E. Archila ◽

M. De LaVega ◽

C. Chen ◽

...

Keyword(s):

Dna Damage ◽

Cell Lines ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Ataxia Telangiectasia ◽

Pancreatic Head ◽

Genotoxic Stress ◽

Ataxia Telangiectasia Mutated ◽

Chromosomal Breakage ◽

Damage Response

10509 Loss of the fanconi anemia (FA) pathway function has been described in a number of sporadic tumor types including breast, ovarian, pancreatic, head and neck and hematological malignancies. Functionally, the FA pathway responds to stalled DNA replication following DNA damage. Given the importance of the FA pathway in the response to DNA damage, we hypothesized that cells deficient in this pathway may become hyper-dependent on alternative DNA damage response pathways in order to respond to endogenous genotoxic stress such as occurs during metabolism. Therefore, targeting these alternative pathways could offer therapeutic strategies in FA pathway deficient tumors. To identify new therapeutic targets we treated FA pathway competent and deficient cells with a DNA damage response siRNA library, that individually knocked out 230 genes. We identified a number of gene targets that were specifically toxic to FA pathway deficient cells, amongst which was the DNA damage response kinase Ataxia Telangiectasia Mutated (ATM). To test the requirement for ATM in FA pathway deficient cells, we interbred Fancg ± Atm± mice. Consistent with the siRNA screen result, Fancg-/- Atm-/- mice were non viable and Fancg± Atm-/- and Fancg-/- Atm ± progeny were less frequent that would have been expected. Several human cell lines with FA gene mutations were observed to have constitutive activation of ATM which was markedly reduced on correction with the appropriate wild-type FA gene. Interestingly, FA pathway deficient cells, including the FANCC mutant and FANCG mutant pancreatic cancer cell lines, were selectively sensitive to monotherapy with the ATM inhibitor KU55933, as measured by dose inhibition and colony count assays. FA pathway deficient cells also demonstrated an increased level of chromosomal breakage, cell cycle arrest and apoptosis following KU55933 treatment when compared to FA pathway corrected cells. We conclude that FA pathway deficient cells have an increased requirement for ATM activation in order to respond to sporadic DNA damage. This offers the possibility that monotherapy with ATM inhibitors could be a therapeutic strategy for tumors that are deficient for the FA pathway. No significant financial relationships to disclose.

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DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response

Nature Communications ◽

10.1038/s41467-019-10408-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

Author(s):

Fengshan Liang ◽

Adam S. Miller ◽

Simonne Longerich ◽

Caroline Tang ◽

David Maranon ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Damage Response

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The interplay of Fanconi anemia proteins in the DNA damage response

DNA Repair ◽

10.1016/j.dnarep.2004.04.005 ◽

2004 ◽

Vol 3 (8-9) ◽

pp. 1063-1069 ◽

Cited By ~ 44

Author(s):

XiaoZhe Wang ◽

Alan D. D’Andrea

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Damage Response

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Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.

Blood ◽

10.1182/blood.v108.11.186.186 ◽

2006 ◽

Vol 108 (11) ◽

pp. 186-186

Author(s):

Natalie B. Collins ◽

Andrei Tomashevski ◽

Gary M. Kupfer

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Phosphorylation Site ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Damage Response ◽

And Function ◽

Mutant Cells

Abstract Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.

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Fanconi Anemia Group J Helicase and MRE11 Nuclease Interact To Facilitate the DNA Damage Response

Molecular and Cellular Biology ◽

10.1128/mcb.01256-12 ◽

2013 ◽

Vol 33 (11) ◽

pp. 2212-2227 ◽

Cited By ~ 36

Author(s):

A. N. Suhasini ◽

J. A. Sommers ◽

P. A. Muniandy ◽

Y. Coulombe ◽

S. B. Cantor ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Damage Response ◽

Anemia Group

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Interaction of the Human Papillomavirus E1 Helicase with UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes

mBio ◽

10.1128/mbio.00152-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 1

Author(s):

Marit Orav ◽

David Gagnon ◽

Jacques Archambault

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Human Papillomaviruses ◽

Viral Genomes ◽

Damage Response ◽

Theta Replication ◽

Infected Cells ◽

Cellular Dna ◽

Medical Burden

ABSTRACTHuman papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCEHuman papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.

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