Abstract
Telomere maintenance is essential for continued cell proliferation. Although most cells accomplish this by activating telomerase, a subset of immortalized tumors and cell lines do so in a telomerase-independent manner, a process called alternative lengthening of telomeres (ALT). DNA recombination has been shown to be involved in ALT, but the precise mechanisms remain unknown. A fraction of cells in a given ALT population contain a unique nuclear structure called APB (ALT-associated promyelocytic leukemia (PML) body), which is characterized by the presence of telomeric DNA in the PML body. Here we describe that hRad9, hHus1, and hRad1, which form a DNA clamp complex that is associated with DNA damage, as well as its clamp loader, hRad17, are constitutive components of APB. Phosphorylated histone H2AX (gamma-H2AX), a molecular marker of double-strand breaks (DSBs), also colocalizes with some APBs. The results suggest that telomeric DNAs at APBs are recognized as DSBs. PML staining and fluorescence in situ hybridization analyses of mitotic ALT cells revealed that telomeric DNAs present at APBs are of both extrachromosomal and native telomere origins. Furthermore, we demonstrated that DNA synthesis occurs at APBs and is significantly inhibited by caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases. Taken together, we suggest that telomeric DNAs at APBs are recognized and processed as DSBs, leading to telomeric DNA synthesis and thereby contributing to telomere maintenance in ALT cells.
Highlights
Telomere maintenance is essential for continued cell proliferation
When total hRad9 foci were examined for telomeric DNA and/or Promyelocytic leukemia (PML) signals, most hRad9 foci in alternative lengthening of telomeres (ALT) cells were double-positive for telomeric DNA and PML, thereby representing APBs
We demonstrated that hRad9, hHus1, hRad1, and hRad17 are localized at APBs in ALT cells
Summary
Proteins involved in DNA metabolism, such as BLM, Mre, and NBS1, are contained in PML bodies (9 –11). NBS1, Mre, and Rad form a complex called the MRN complex, which plays a critical role in DNA damage sensing, repair, and checkpoint. These results suggest that PML bodies are involved in genomic stability. 5-bromo-2Ј-deoxyuridine (BrdUrd) incorporation was detected at the NBS1 foci in a small fraction of growing ALT cells, suggesting that DNA is synthesized at APBs [15]. We present evidence that the DNA damage checkpoint is operating in the APB-positive ALT cells
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