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

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

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

The CHD4 helicase is identified as a new component of the genome surveillance machinery in a proteomic screen for factors enriched on chromatin after ionizing radiation (see also related paper by Smeenk et al. in this issue)

Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease

Wee1 is essential for normal DNA replication and for genomic stability, at least in part by inhibiting a general DNA damage response induced by the Mus81-Eme1 endonuclease

DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by the RNF8/RNF168/HERC2 ubiquitin ligases facilitates restoration of genome integrity by licensing chromatin to concentrate genome caretaker proteins near the lesions. In parallel, SUMOylation of so-far elusive upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response. We show that HERC2 and RNF168 are novel DNA damage–dependent SUMOylation targets in human cells. In response to DSBs, both HERC2 and RNF168 were specifically modified with SUMO1 at DSB sites in a manner dependent on the SUMO E3 ligase PIAS4. SUMOylation of HERC2 was required for its DSB-induced association with RNF8 and for stabilizing the RNF8–Ubc13 complex. We also demonstrate that the ZZ Zinc finger in HERC2 defined a novel SUMO-specific binding module, which together with its concomitant SUMOylation and T4827 phosphorylation promoted binding to RNF8. Our findings provide novel insight into the regulatory complexity of how ubiquitylation and SUMOylation cooperate to orchestrate protein interactions with DSB repair foci

A Novel Role of Human Holliday Junction Resolvase GEN1 in the Maintenance of Centrosome Integrity

<div><p>The maintenance of genomic stability requires accurate genome replication, repair of DNA damage, and the precise segregation of chromosomes in mitosis. GEN1 possesses Holliday junction resolvase activity <em>in vitro</em> and presumably functions in homology driven repair of DNA double strand breaks. However, little is currently known about the cellular functions of human GEN1. In the present study we demonstrate that GEN1 is a novel centrosome associated protein and we characterize the various phenotypes associated with GEN1 deficiency. We identify an N-terminal centrosome localization signal in GEN1, which is required and sufficient for centrosome localization. We report that GEN1 depletion results in aberrant centrosome numbers associated with the formation of multiple spindle poles in mitosis, an increased number of cells with multi-nuclei, increased apoptosis and an elevated level of spontaneous DNA damage. We find homologous recombination severely impaired in GEN1 deficient cells, suggesting that GEN1 functions as a Holliday junction resolvase <em>in vivo</em> as well as <em>in vitro</em>. Complementation of GEN1 depleted cells with various GEN1 constructs revealed that centrosome association but not catalytic activity of GEN1 is required for preventing centrosome hyper-amplification, formation of multiple mitotic spindles, and multi-nucleation. Our findings provide novel insight into the biological functions of GEN1 by uncovering an important role of GEN1 in the regulation of centrosome integrity.</p> </div

GEN1 depletion interferes with cell cycle progression and results in multi-nucleation and apoptosis. A.

<p>(left) 293T cells were treated with CTRL or GEN1 siRNA for 48 h stained with propidium iodine (PI) and analyzed by flow cytometry. (middle) G2/M cells were identified using Modfit software. (right) Mitotic cells were detected by phospho H3 antibody staining followed by flow cytometric analysis. Quantification is based on three independent experiments. Error bars indicate S.E.M. <b>B.</b> Hela cells were treated with CTRL or GEN1 siRNA for 48 h, labeled with or without 10 uM BrdU for 2 hours, stained with FITC conjugated anti-BrdU antibodies and propidium iodine (PI), and analyzed by flow cytometry. x-axis: propidium iodine (PI). y-axis FITC. <b>C.</b> CTRL or GEN1 siRNA treated Hela cells were incubated with 10 uM BrdU for 2 hours, and return to BrdU-free medium for the indicated times. Cell cycle progression of BrdU labeled cells were analyzed by flow cytometry. <b>D.</b> HeLa cells treated with CTRL or GEN1 siRNA (48 h) were methanol fixed and immunostained with GEN1(anti-GEN1 (651–892aa) antibody) and α-tubulin. DNA was visualized by DAPI. (Right) Graph represents the mean of three independent experiments. (n = 100 cells/condition/experiment). Error bars indicate S.E.M. <b>E.</b> HeLa cells were treated with CTRL or GEN1 siRNA for 48 h fixed, stained with annexin V and analyzed by flow cytometry. Quantification was based on three independent experiments. Error bars indicate S.E.M.</p