Abstract
The ATR checkpoint pathway responds to DNA damage during the S/G2 phases of the cell cycle and is activated early in tumorigenesis. Investigation of ATR’s role in development and tumorigenesis is complicated by the lethality of homozygous knockout mice and the limited effects of heterozygous deficiency. To overcome this limitation, we sought to create mice with a hypomorphic Atr mutation based on the ATR mutation in the human disease Seckel syndrome-1 (SCKL1). Homozygous SCKL1 mice were generated by targeted knock-in of the A → G SCKL1 mutation. Western blot and RT-PCR analysis established that homozygotes have no reduction in Atr protein or increase in missplicing as is seen in humans. Thus, the A → G substitution alone is not sufficient to reproduce in mice the effects that are seen in humans. However, homozygous SCKL1 mice that retain the neo cassette used for targeting have an estimated 66-82% reduction in total Atr protein levels due to missplicing into the neo cassette. Under conditions of APH-induced replication stress, primary fibroblasts from homozygous mice displayed an increase in overall chromosome damage and an increase in gaps and breaks at specific common fragile sites. In addition, mutant cells display a significant delay in checkpoint induction and an increase in DNA damage as assayed by Chk1 phosphorylation and γ-H2ax levels, respectively. These mice provide a novel model system for studies of Atr deficiency and replication stress.
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Acknowledgments
We thank Sally Camper and David Ferguson for their assistance and advice in critically reading the manuscript. We thank Matthew Butler for his time and technical assistance. This work was made possible by support from the National Institutes of Health grant CA43222 and the John and Suzanne Munn Research Fund from the University of Michigan Comprehensive Cancer Center.
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Supplementary Fig. 1
Splicing pattern observed by RT-PCR from normal mouse total mRNA.(A) PCR scheme used for detection of misspliced products lacking exon 9. Primers used are labeled p4 and p5. (B) Agarose gel showing both normal and misspliced product lacking exon 9. (C) Junction sequences between exons 8, 9, and 10 in the normal splice form and exons 8 and 10 in the misspliced form lacking exon 9 (TIFF 1934 kb)
Supplementary Fig. 2
A representative partial metaphase spread from AtrSckN/SckN tail fibroblasts with FISH probes to the common fragile sites Fra14A2 and Fra8E1 shown in red and green, respectively. Both FRA14A2 loci show fragile site breaks and the leftmost Fra8E1 locus is broken in this example (TIFF 2300 kb)
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Ragland, R.L., Arlt, M.F., Hughes, E.D. et al. Mice hypomorphic for Atr have increased DNA damage and abnormal checkpoint response. Mamm Genome 20, 375–385 (2009). https://doi.org/10.1007/s00335-009-9195-4
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DOI: https://doi.org/10.1007/s00335-009-9195-4