Abstract
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.
Highlights
Nuclear substructures, known as promyelocytic leukemia (PML) nuclear bodies (NBs), PML oncogenic domains (PODs) or nuclear domain 10 (ND10), are associated with nuclear matrix and play important roles in diverse cellular processes such as transcription, chromatin dynamics, oncogenesis, posttranslational modifications, apoptosis, p53 regulation, senescence, DNA damage repair and antiviral responses [1,2,3,4]
We found that in large T antigen (LT)-expressing cells, Rad51 foci depend on PML
The emphasis here was on Rad51, PML clearly plays a role in localizing, or otherwise regulating, additional DDR factors such as TopBP1, BLM, MRN, Rockland (pATM S1981) and Neomarkers (RPA) and BRCA1 ([12,13], Fig. 5a)
Summary
Known as promyelocytic leukemia (PML) nuclear bodies (NBs), PML oncogenic domains (PODs) or nuclear domain 10 (ND10), are associated with nuclear matrix and play important roles in diverse cellular processes such as transcription, chromatin dynamics, oncogenesis, posttranslational modifications, apoptosis, p53 regulation, senescence, DNA damage repair and antiviral responses [1,2,3,4]. PML is the nucleating component of PML NBs, essential for their formation, and loss of PML results in dispersal of the other components residing at PML NBs. There is known to be a rapid exchange between the PML NBs and the nucleoplasm [1]. There are constitutive components of PML NBs such as PML, DAXX and Sp100 and a large number (.50) of proteins that can transiently associate with PML NBs, for example p53 and p300/CBP [1]. PML, as well as several other PML NB components, are modified by small ubiquitin-like modifier (SUMO) and for PML this is essential for formation and maintenance of the PML NBs [6,7]
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