Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities

Homologous recombination (HR) is essential for faithful repair of DNA lesions yet must be kept in check, as unrestrained HR may compromise genome integrity and lead to premature aging or cancer. To limit unscheduled HR, cells possess DNA helicases capable of preventing excessive recombination. In this study, we show that the human Fbh1 (hFbh1) helicase accumulates at sites of DNA damage or replication stress in a manner dependent fully on its helicase activity and partially on its conserved F box. hFbh1 interacted with single-stranded DNA (ssDNA), the formation of which was required for hFbh1 recruitment to DNA lesions. Conversely, depletion of endogenous Fbh1 or ectopic expression of helicase-deficient hFbh1 attenuated ssDNA production after replication block. Although elevated levels of hFbh1 impaired Rad51 recruitment to ssDNA and suppressed HR, its small interfering RNA–mediated depletion increased the levels of chromatin-associated Rad51 and caused unscheduled sister chromatid exchange. Thus, by possessing both pro- and anti-recombinogenic potential, hFbh1 may cooperate with other DNA helicases in tightly controlling cellular HR activity.

Download Full-text

A Role for Fis1 in Both Mitochondrial and Peroxisomal Fission in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0159 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5077-5086 ◽

Cited By ~ 198

Author(s):

Annett Koch ◽

Yisang Yoon ◽

Nina A. Bonekamp ◽

Mark A. McNiven ◽

Michael Schrader

Keyword(s):

Mammalian Cells ◽

Small Interfering Rna ◽

Transmembrane Domain ◽

Mitochondrial Fission ◽

Ectopic Expression ◽

Mammalian Homologue ◽

Necessary And Sufficient ◽

Interfering Rna ◽

Peroxisome Division

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.

Download Full-text

Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

The Journal of Cell Biology ◽

10.1083/jcb.200905059 ◽

2010 ◽

Vol 188 (5) ◽

pp. 629-638 ◽

Cited By ~ 112

Author(s):

Halfdan Beck ◽

Viola Nähse ◽

Marie Sofie Yoo Larsen ◽

Petra Groth ◽

Trevor Clancy ◽

...

Keyword(s):

Dna Damage ◽

Human Cell Line ◽

S Phase ◽

Dna Lesions ◽

Genome Integrity ◽

Replication Proteins ◽

Cyclin Dependent Kinases ◽

Mitotic Kinase ◽

Cellular Control ◽

Interfering Rna

Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication.

Download Full-text

Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains

Molecular and Cellular Biology ◽

10.1128/mcb.00166-09 ◽

2009 ◽

Vol 29 (10) ◽

pp. 2570-2581 ◽

Cited By ~ 787

Author(s):

Grégory Bellot ◽

Raquel Garcia-Medina ◽

Pierre Gounon ◽

Johanna Chiche ◽

Danièle Roux ◽

...

Keyword(s):

Cell Death ◽

Tumor Progression ◽

Cell Survival ◽

Small Interfering Rna ◽

Hypoxia Inducible Factor ◽

Ectopic Expression ◽

Survival Mechanism ◽

Hypoxic Conditions ◽

Interfering Rna

ABSTRACT While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy, enhances cell death under hypoxic conditions. In addition, the ablation of BNIP3 and/or BNIP3L triggers cell death, and BNIP3 and BNIP3L are crucial for hypoxia-induced autophagy. First, while the small interfering RNA-mediated ablation of either BNIP3 or BNIP3L has little effect on autophagy, the combined silencing of these two HIF targets suppresses hypoxia-mediated autophagy. Second, the ectopic expression of both BNIP3 and BNIP3L in normoxia activates autophagy. Third, 20-mer BH3 peptides of BNIP3 or BNIP3L are sufficient in initiating autophagy in normoxia. Herein, we propose a model in which the atypical BH3 domains of hypoxia-induced BNIP3/BNIP3L have been designed to induce autophagy by disrupting the Bcl-2-Beclin1 complex without inducing cell death. Hypoxia-induced autophagy via BNIP3 and BNIP3L is clearly a survival mechanism that promotes tumor progression.

Download Full-text