This Article Full Text Full Text (PDF) Other Versions of this Article: MCB.01763-06v1 27/6/2189    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Levitt, P. S. Articles by Weiss, R. S. Search for Related Content PubMed PubMed Citation Articles by Levitt, P. S. Articles by Weiss, R. S.

Genome Maintenance Defects in Cultured Cells and Mice following Partial Inactivation of the Essential Cell Cycle Checkpoint Gene Hus1

Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853

Received 18 September 2006/ Returned for modification 11 October 2006/ Accepted 20 December 2006

Cell cycle checkpoints are evolutionarily conserved signaling pathways that uphold genomic integrity. Complete inactivation of the mouse checkpoint gene Hus1 results in chromosomal instability, genotoxin hypersensitivity, and embryonic lethality. To determine the functional consequences of partial Hus1 impairment, we generated an allelic series in which Hus1 expression was incrementally reduced by combining a hypomorphic Hus1 allele, Hus1^neo, with either wild-type or null (Hus1¹) alleles. Primary Hus1^neo/1 embryonic fibroblasts exhibited spontaneous chromosomal abnormalities and underwent premature senescence, while higher Hus1 expression in Hus1^neo/neo cells allowed for normal proliferation. Antioxidant treatment almost fully suppressed premature senescence in Hus1^neo/1 cultures, suggesting a critical role for Hus1 in oxidative stress responses. Treatment of Hus1^neo/neo and Hus1^neo/1 cells with the DNA adducting agent benzo(a)pyrene dihydrodriol epoxide resulted in a loss of cell viability that was associated with S-phase DNA damage checkpoint failure. Likewise, the DNA polymerase inhibitor aphidicolin triggered increased cell death, chromosomal aberrations, and H2AX phosphorylation, a marker for double-stranded DNA breaks, in Hus1^neo/neo and Hus1^neo/1 cultures compared to controls. Despite these pronounced genome maintenance defects in cultured Hus1^neo/1 and Hus1^neo/neo cells, mice of the same genotypes were born at expected frequencies and appeared grossly normal. A significant increase in micronucleus formation was observed in peripheral blood cells from Hus1^neo/1 mice, but reduced Hus1 expression did not cause an elevated predisposition to spontaneous tumor development or accelerate tumorigenesis in p53-deficient mice. These results identify differential effects of altered Hus1 gene dosage on genome maintenance during in vitro culture, genotoxic stress responses, embryonic development, and adult homeostasis.

Published ahead of print on 12 January 2007.