Abstract
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 in vitro 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 in vivo as well as in vitro. 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.
Highlights
DNA double strand breaks (DSBs) represent some of the most cytotoxic lesions and pose a major threat to genome stability if not properly repaired [1,2,3]
According to immunostainings of exponentially growing MRC5 cells captured in interphase and at various stages of mitosis, the centrosomal localization of GEN1 is not regulated during the cell cycle (Fig. 1C)
This would be inconsistent with previous studies and since they display the same specific enzymatic activity towards static HJs in vitro we find this unlikely [16,17,22]. We believe that it either reflects the existence of residual protein masking any additional effects or that the loss of the HJ resolvases is compensated for by increased activity of the BLM-TopIII and/or Mus81-Eme1 complex in siGEN1/siSLX4 cells. It may be explained by the existence of a yet not identified protein involved in the resolution/dissolution of HJs, which can act redundantly with GEN1 and SLX1-SLX4
Summary
DNA double strand breaks (DSBs) represent some of the most cytotoxic lesions and pose a major threat to genome stability if not properly repaired [1,2,3]. DSBs are primarily repaired by two mechanisms: non-homologous end joining and homologous recombination (HR) [4,5]. Following recognition and initial resection of the DSB by the MRN (Mre11–Rad50–NBS1) complex, broken ends are further resected by BLM and EXO1 to generate 39 single-strand overhangs [6,7,8]. Migration of the D-loop results in capture of the second end of the DSB and subsequent ligation leads to the formation of the double Holliday junction (dHJ) structure. Classical HJ resolving enzymes are nucleases that resolve dHJs by introducing two perfectly symmetrical cleavages that result in either crossover or non-crossover products, depending on the orientation of the cleavage site. Deletion of yen in budding yeast resulted in no obvious DNA repair defect, but when combined with deletion of
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