Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression.

Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.

Partial reconstitution of hepatitis C virus RNA polymerization by heterologous expression of NS5B polymerase and template RNA in bacterial cell.

The hepatitis C virus (HCV) is a major etiological agent causing chronic hepatitis in humans. Since the virus does not grow in a cell culture, the direct measurement of viral replication is impossible. Therefore, the current study presents a surrogate model system using a viral polymerase and RNA template. A plasmid expressing the HCV NS5B polymerase was maintained with a plasmid containing a reporter gene in an Escherichia coli cell. The reporter construct contained the HCV 5' untranslated region (UTR) followed by a luciferase gene with a specific orientation so that a minus-sense transcript containing the luciferase fused to the 5' UTR was produced after the initial transcription. When the HCV NS5B polymerase was expressed in the same cell, the primary transcript was recognized by the polymerase due to the presence of the minus-sense 5' UTR, and a secondary transcript containing a plus-sense luciferase gene was produced. Thus, a simple luciferase assay was able to measure the HCV NS5B polymerase activity. The production of minus- and plus-sense transcripts was confirmed by an RT-PCR, while the production of HCV NS5B and expression of the reporter luciferase in the bacterial cell were confirmed by immunofluorescence microscopy. The polymerization occurred in the absence of any other viral/host factors. Accordingly, this would appear to be the first study to demonstrate that the heterologous expression of an animal viral RNA polymerase and its template in a bacterial cell can partially reconstitute the polymerization reaction.