JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
ATR phosphorylates SMARCAL1 to prevent replication fork collapse.
Genes Dev.
PUBLISHED: 07-23-2013
Show Abstract
Hide Abstract
The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.
Related JoVE Video
Identification and characterization of SMARCAL1 protein complexes.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
SMARCAL1 is an ATPase in the SNF2 family that functions at damaged replication forks to promote their stability and restart. It acts by translocating on DNA to catalyze DNA strand annealing, branch migration, and fork regression. Many SNF2 enzymes work as motor subunits of large protein complexes. To determine if SMARCAL1 is also a member of a protein complex and to further understand how it functions in the replication stress response, we used a proteomics approach to identify interacting proteins. In addition to the previously characterized interaction with replication protein A (RPA), we found that SMARCAL1 forms complexes with several additional proteins including DNA-PKcs and the WRN helicase. SMARCAL1 and WRN co-localize at stalled replication forks independently of one another. The SMARCAL1 interaction with WRN is indirect and is mediated by RPA acting as a scaffold. SMARCAL1 and WRN act independently to prevent MUS81 cleavage of the stalled fork. Biochemical experiments indicate that both catalyze fork regression with SMARCAL1 acting more efficiently and independently of WRN. These data suggest that RPA brings a complex of SMARCAL1 and WRN to stalled forks, but that they may act in different pathways to promote fork repair and restart.
Related JoVE Video
Thr-1989 phosphorylation is a marker of active ataxia telangiectasia-mutated and Rad3-related (ATR) kinase.
J. Biol. Chem.
PUBLISHED: 06-24-2011
Show Abstract
Hide Abstract
The DNA damage response kinases ataxia telangiectasia-mutated (ATM), DNA-dependent protein kinase (DNA-PK), and ataxia telangiectasia-mutated and Rad3-related (ATR) signal through multiple pathways to promote genome maintenance. These related kinases share similar methods of regulation, including recruitment to specific nucleic acid structures and association with protein activators. ATM and DNA-PK also are regulated via phosphorylation, which provides a convenient biomarker for their activity. Whether phosphorylation regulates ATR is unknown. Here we identify ATR Thr-1989 as a DNA damage-regulated phosphorylation site. Selective inhibition of ATR prevents Thr-1989 phosphorylation, and phosphorylation requires ATR activation. Cells engineered to express only a non-phosphorylatable T1989A mutant exhibit a modest ATR functional defect. Our results suggest that, like ATM and DNA-PK, phosphorylation regulates ATR, and phospho-peptide specific antibodies to Thr-1989 provide a proximal marker of ATR activation.
Related JoVE Video
Functional genomic screens identify CINP as a genome maintenance protein.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-04-2009
Show Abstract
Hide Abstract
The DNA damage response (DDR) has a critical role in maintaining genome integrity and serves as a barrier to tumorigenesis by promoting cell-cycle arrest, DNA repair, and apoptosis. The DDR is activated not only by genotoxic agents that induce DNA damage, but also during aberrant cell-division cycles caused by activated oncogenes and inactivated tumor suppressors. Here we use RNAi and cDNA overexpression screens in human cells to identify genes that, when deregulated, lead to activation of the DDR. The RNAi screen identified 73 genes that, when silenced in at least two cell types, cause DDR activation. Silencing several of these genes also caused an increased frequency of micronuclei, a marker of genetically unstable cells. The cDNA screen identified 97 genes that when overexpressed induce DDR activation in the absence of any exogenous genotoxic agent, with an overrepresentation of genes linked to cancer. Secondary RNAi screens identified CDK2-interacting protein (CINP) as a cell-cycle checkpoint protein. CINP interacts with ATR-interacting protein and regulates ATR-dependent signaling, resistance to replication stress, and G2 checkpoint integrity.
Related JoVE Video
The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks.
Genes Dev.
PUBLISHED: 09-30-2009
Show Abstract
Hide Abstract
Mutations in SMARCAL1 (HARP) cause Schimke immunoosseous dysplasia (SIOD). The mechanistic basis for this disease is unknown. Using functional genomic screens, we identified SMARCAL1 as a genome maintenance protein. Silencing and overexpression of SMARCAL1 leads to activation of the DNA damage response during S phase in the absence of any genotoxic agent. SMARCAL1 contains a Replication protein A (RPA)-binding motif similar to that found in the replication stress response protein TIPIN (Timeless-Interacting Protein), which is both necessary and sufficient to target SMARCAL1 to stalled replication forks. RPA binding is critical for the cellular function of SMARCAL1; however, it is not necessary for the annealing helicase activity of SMARCAL1 in vitro. An SIOD-associated SMARCAL1 mutant fails to prevent replication-associated DNA damage from accumulating in cells in which endogenous SMARCAL1 is silenced. Ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) phosphorylate SMARCAL1 in response to replication stress. Loss of SMARCAL1 activity causes increased RPA loading onto chromatin and persistent RPA phosphorylation after a transient exposure to replication stress. Furthermore, SMARCAL1-deficient cells are hypersensitive to replication stress agents. Thus, SMARCAL1 is a replication stress response protein, and the pleiotropic phenotypes of SIOD are at least partly due to defects in genome maintenance during DNA replication.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.