Bloom’s Syndrome and PICH Helicases Cooperate with Topoisomerase IIα in Centromere Disjunction before Anaphase

Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges

Functional Connection between Rad51 and PML in Homology-Directed Repair

The promyelocytic leukemia protein (PML) is a tumor suppressor critical for formation of nuclear bodies (NBs) performing important functions in transcription, apoptosis, DNA repair and antiviral responses. Earlier studies demonstrated that simian virus 40 (SV40) initiates replication near PML NBs. Here we show that PML knockdown inhibits viral replication in vivo, thus indicating a positive role of PML early in infection. SV40 large T antigen (LT) induces DNA damage and, consequently, nuclear foci of the key homologous recombination repair protein Rad51 that colocalize with PML. PML depletion abrogates LT-induced Rad51 foci. LT may target PML NBs to gain access to DNA repair factors like Rad51 that are required for viral replication. We have used the SV40 model to gain insight to DNA repair events involving PML. Strikingly, even in normal cells devoid of viral oncoproteins, PML is found to be instrumental for foci of Rad51, Mre11 and BRCA1, as well as homology-directed repair after double-strand break (DSB) induction. Following LT expression or external DNA damage, PML associates with Rad51. PML depletion also causes a loss of RPA foci following γ-irradiation, suggesting that PML is required for processing of DSBs. Immunofluorescent detection of incorporated BrdU without prior denaturation indicates a failure to generate ssDNA foci in PML knockdown cells upon γ-irradiation. Consistent with the lack of RPA and BrdU foci, γ-irradiation fails to induce Chk1 activation, when PML is depleted. Taken together, we have discovered a novel functional connection between PML and the homologous recombination-mediated repair machinery, which might contribute to PML tumor suppressor activity

SUMO Interaction Motifs in Sizn1 Are Required for Promyelocytic Leukemia Protein Nuclear Body Localization and for Transcriptional Activation*

Mutations in Sizn1 (Zcchc12), a novel transcriptional co-activator in the BMP signaling pathway, are associated with X-linked mental retardation. Previously, we demonstrated that Sizn1 positively modulates the BMP signal by interacting with Smad family members and cAMP-responsive element-binding protein-binding protein. To further define the molecular basis of Sizn1 function, we have explored its subcellular localization and generated various deletion mutants to carry out domain analyses. Here, we report that Sizn1 localizes to promyelocytic leukemia protein nuclear bodies (PML-NBs). Sizn1 deletion mutants that disrupt the MA homologous domain or the middle region fail to target to the PML-NB. We show that two SUMO interaction motifs (SIMs) in Sizn1 can bind to SUMO and govern SUMO conjugation to Sizn1 in the absence of the consensus motif for SUMO attachment. Interestingly, the SIM mutant Sizn1 localizes to nuclear bodies, but not to PML-NBs. Thus, SIMs mediate the localization of Sizn1 to PML-NB. Interestingly, mutations in SIM sequences and deletion of the MA homologous domain also affected the transcriptional co-activation function of a Sizn1. Taken together, our data indicate that the SIMs in Sizn1 are required for its PML-NB localization and for the full transcriptional co-activation function in BMP signaling

Activation of the SUMO modification system is required for the accumulation of RAD51 at sites containing DNA damage

Genetic information encoded in chromosomal DNA is challenged by intrinsic and exogenous sources of DNA damage. DNA double-strand 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

A caspase-3-cleaved fragment of the glial glutamate transporter EAAT2 is sumoylated and targeted to promyelocytic leukemia nuclear bodies in mutant SOD1-linked amyotrophic lateral sclerosis

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na +-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS. \ua9 2007 by The American Society for Biochemistry and Molecular Biology, Inc