Mice hypomorphic for Atr have increased DNA damage and abnormal checkpoint response.
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 gamma-H2ax levels, respectively. These mice provide a novel model system for studies of Atr deficiency and replication stress.
Ragland
Ryan L
RL
Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-0618, USA. ryanragland@yahoo.com
Arlt
Martin F
MF
Hughes
Elizabeth D
ED
Saunders
Thomas L
TL
Glover
Thomas W
TW
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004195:Disease Models, Animal; D004392:Dwarfism / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism
D018797:Cell Cycle Proteins; C576897:Atr protein, mouse; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases