Increased common fragile site expression, cell proliferation defects, and apoptosis following conditional inactivation of mouse Hus1 in primary cultured cells

Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.
Molecular Biology of the Cell (Impact Factor: 4.55). 04/2007; 18(3):1044-55. DOI: 10.1091/mbc.E06-10-0957
Source: PubMed

ABSTRACT Targeted disruption of the mouse Hus1 cell cycle checkpoint gene results in embryonic lethality and proliferative arrest in cultured cells. To investigate the essential functions of Hus1, we developed a system for the regulated inactivation of mouse Hus1 in primary fibroblasts. Inactivation of a loxP site-flanked conditional Hus1 allele by using a cre-expressing adenovirus resulted in reduced cell doubling, cell cycle alterations, and increased apoptosis. These phenotypes were associated with a significantly increased frequency of gross chromosomal abnormalities and an S-phase-specific accumulation of phosphorylated histone H2AX, an indicator of double-stranded DNA breaks. To determine whether these chromosomal abnormalities occurred randomly or at specific genomic regions, we assessed the stability of common fragile sites, chromosomal loci that are prone to breakage in cells undergoing replication stress. Hus1 was found to be essential for fragile site stability, because spontaneous chromosomal abnormalities occurred preferentially at common fragile sites upon conditional Hus1 inactivation. Although p53 levels increased after Hus1 loss, deletion of p53 failed to rescue the cell-doubling defect or increased apoptosis in conditional Hus1 knockout cells. In summary, we propose that Hus1 loss leads to chromosomal instability during DNA replication, triggering increased apoptosis and impaired proliferation through p53-independent mechanisms.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The WRN protein belongs to the RecQ family of DNA helicases and is implicated in replication fork restart, but how its function is regulated remains unknown. We show that WRN interacts with the 9.1.1 complex, one of the central factors of the replication checkpoint. This interaction is mediated by the binding of the RAD1 subunit to the N-terminal region of WRN and is instrumental for WRN relocalization in nuclear foci and its phosphorylation in response to replication arrest. We also find that ATR-dependent WRN phosphorylation depends on TopBP1, which is recruited by the 9.1.1 complex in response to replication arrest. Finally, we provide evidence for a cooperation between WRN and 9.1.1 complex in preventing accumulation of DNA breakage and maintaining genome integrity at naturally occurring replication fork stalling sites. Taken together, our data unveil a novel functional interplay between WRN helicase and the replication checkpoint, contributing to shed light into the molecular mechanism underlying the response to replication fork arrest.
    Oncogene 10/2011; 31(23):2809-23. DOI:10.1038/onc.2011.468 · 8.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Chromosome rearrangements such as translocations and deletions are frequently associated with human cancers. Such rearrangement of the chromosome can be initiated by a DNA break (DSB) that, when inappropriately repaired, may alter chromosome structure. Mammalian common fragile sites are the best-characterised, naturally occurring breakage-prone regions and are deleted or rearranged in many tumour cells. Analogous chromosomal regions also exist in the budding yeast, S. cerevisiae. One example of a yeast fragile site is the replication slow zone (RSZ), so called because the rate of replication fork progression through these regions is slow compared to other regions within the same chromosome. Inactivation of the essential checkpoint kinase, Mec1, in mec1-ts mutants results in replication fork stalling followed by chromosome breakage at RSZs. Interestingly, inhibition of ATR, the mammalian homologue of Mec1, also leads to chromosome instability at common fragile sites, suggesting that the mechanism by which endogenous DSBs are generated is conserved between yeast and mammals. This study aims to enhance our current understanding of common fragile sites using yeast RSZs as a model. First, RSZs were characterised in terms of chromosomal features and determinants in order to identify similarities between RSZs and mammalian common fragile sites and to assess whether yeast RSZs as a suitable system for studying common fragile sites in more complex organisms. Next, the mechanism underlying chromosome fragility at RSZs was investigated by examining the contribution of various chromosomal processes to break formation at these sites. These include: (i) replication fork restart processes (ii) spindle force, (iii) chromosome condensation and decatenation, (iv) chromosome segregation, and (v) cytokinesis. The analyses suggest that chromosome breakage within RSZs requires the actions of the evolutionarily conserved type II topoisomerase and condensin complex. Finally, factors involved in maintaining the stability of RSZs were also explored. The Rrm3 helicase and Psy2 phosphatase complex were found to suppress chromosome breakage at RSZs in a manner dependent on Tel1, another checkpoint kinase. These findings suggest that Tel1 is somehow implicated in chromosome stability at RSZs. The findings presented in this study further our understanding of RSZs and the molecular bases governing their fragility, providing some insight into the mechanism of fragile site instability in mammals.
  • Source


Available from

Similar Publications