Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR) causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.
SMARCAL1 promotes the repair and restart of damaged replication forks. Either overexpression or silencing SMARCAL1 causes the accumulation of replication-associated DNA damage. SMARCAL1 is heavily phosphorylated. Here we identify multiple phosphorylation sites, including S889, which is phosphorylated even in undamaged cells. S889 is highly conserved through evolution and it regulates SMARCAL1 activity. Specifically, S889 phosphorylation increases the DNA-stimulated ATPase activity of SMARCAL1 and increases its ability to catalyze replication fork regression. A phosphomimetic S889 mutant is also hyperactive when expressed in cells, while a non-phosphorylatable mutant is less active. S889 lies within a C-terminal region of the SMARCAL1 protein. Deletion of the C-terminal region also creates a hyperactive SMARCAL1 protein suggesting that S889 phosphorylation relieves an auto-inhibitory function of this SMARCAL1 domain. Thus, S889 phosphorylation is one mechanism by which SMARCAL1 activity is regulated to ensure the proper level of fork remodeling needed to maintain genome integrity during DNA synthesis.
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.
ATR (ataxia telangiectasia-mutated and Rad3-related) contains 16 conserved candidate autophosphorylation sites that match its preferred S/TQ consensus. To determine whether any is functionally important, we mutated the 16 candidate residues to alanine in a single cDNA to create a 16A-ATR mutant. The 16A-ATR mutant maintains kinase and G(2) checkpoint activities. However, it fails to rescue the essential function of ATR in maintaining cell viability and fails to promote replication recovery from a transient exposure to replication stress. Further analysis identified T1566A/T1578A/T1589A (3A-ATR) as critical mutations causing this separation of function activity. Secondary structure predictions indicate that these residues occur in a region between ATR HEAT repeats 31R and 32R that aligns with regions of ATM and DNA-PK containing regulatory autophosphorylation sites. Although this region is important for ATR function, the 3A-ATR residues do not appear to be sites of autophosphorylation. Nevertheless, our analysis identifies an important regulatory region of ATR that is shared among the PI3K-related protein kinase family. Furthermore, our data indicate that the essential function of ATR for cell viability is linked to its function in promoting proper replication in the context of replication stress and is independent of G(2) checkpoint activity.
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.
Cyclin-dependent kinase 9 (CDK9) is a well-characterized subunit of the positive transcription elongation factor b complex in which it regulates transcription elongation in cooperation with cyclin T. However, CDK9 also forms a complex with cyclin K, the function of which is less clear. Using a synthetic lethal RNA interference screen in human cells, we identified CDK9 as a component of the replication stress response. Loss of CDK9 activity causes an increase in spontaneous levels of DNA damage signalling in replicating cells and a decreased ability to recover from a transient replication arrest. This activity is restricted to CDK9-cyclin K complexes and is independent of CDK9-cyclin T complex. CDK9 accumulates on chromatin in response to replication stress and limits the amount of single-stranded DNA in cells under stress. Furthermore, we show that CDK9 and cyclin K interact with ataxia telangiectasia and Rad3-related protein and other checkpoint signalling proteins. These results reveal an unexpectedly direct role for CDK9-cyclin K in checkpoint pathways that maintain genome integrity in response to replication stress.
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
Journal of Visualized Experiments
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.