Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1α), and other RDM1 isoforms. We show that RDM1α undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response

ATM Modulates the Loading of Recombination Proteins onto a Chromosomal Translocation Breakpoint Hotspot

Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots

A role for RAD54B in homologous recombination in human cells

In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans

Haploinsufficiency of the Mus81.Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells

The Mus81.Eme1 complex is a structure-specific endonuclease that preferentially cleaves nicked Holliday junctions, 3'-flap structures and aberrant replication fork structures. Mus81-/- mice have been shown to exhibit spontaneous chromosomal aberrations and, in one of two models, a predisposition to cancers. The molecular mechanisms underlying its role in chromosome integrity, however, are largely unknown. To clarify the role of Mus81 in human cells, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here we demonstrate that Mus81 confers resistance to DNA crosslinking agents and slight resistance to other DNA-damaging agents. Mus81 deficiency spontaneously promotes chromosome damage such as breaks and activates the intra-S-phase checkpoint through the ATM-Chk1/Chk2 pathways. Furthermore, Mus81 deficiency activates the G2/M checkpoint through the ATM-Chk2 pathway and promotes DNA rereplication. Increased rereplication is reversed by the ectopic expression of Cdk1. Haploinsufficiency of Mus81 orEme1 also causes similar phenotypes.These findings suggest that a complex network of the checkpoint pathways that respond to DNA doublestrand breaks may participate in some of the phenotypes associated with Mus81 or Eme1 deficiency

Il piacere di imparare il piacere di insegnare

textabstractGenetic information encoded in chromosomal DNA is challenged by intrinsic and exogenous sources of DNA damage. DNA doublestrand breaks (DSBs) are extremely dangerous DNA lesions. RAD51 plays a central role in homologous DSB repair, by facilitating the recombination of damaged DNA with intact DNA in eukaryotes. RAD51 accumulates at sites containing DNA damage to form nuclear foci. However, the mechanism of RAD51 accumulation at sites of DNA damage is still unclear. Post-translational modifications of proteins, such as phosphorylation, acetylation and ubiquitylation play a role in the regulation of protein localization and dynamics. Recently, the covalent binding of small ubiquitin-like modifier (SUMO) proteins to target proteins, termed SUMOylation, at sites containing DNA damage has been shown to play a role in the regulation of the DNA-damage response. Here, we show that the SUMOylation E2 ligase UBC9, and E3 ligases PIAS1 and PIAS4, are required for RAD51 accretion at sites containing DNA damage in human cells. Moreover, we identified a SUMO-interacting motif (SIM) in RAD51, which is necessary for accumulation of RAD51 at sites of DNA damage. These findings suggest that the SUMO-SIM system plays an important role in DNA repair, through the regulation of RAD51 dynamics