Promyelocytic nuclear bodies (PML-NBs) are distinct nuclear structures that are involved in apoptosis, differentiation, transcriptional regulation and DNA damage response. These bodies have also been shown to associate with nuclear sites of viral DNA replication. In the present study, we used BrdU pulse labeling to demonstrate that PML-NBs accumulate newly synthesized DNA in cells infected by the polyomaviruses simian virus 40 (SV40) or polyomavirus BK (BKV). Sequestration of DNA molecules in these structures depended on active viral DNA replication, and was observed exclusively in cells that contained prominent viral replication domains. Furthermore, a significant portion of the accumulated DNA was found to be single-stranded, indicating that the sequestered DNA had been subjected to processing by nuclease or DNA unwinding activities. siRNA-mediated suppression of the PML protein prevented the recruitment of single-stranded DNA into nuclear foci, but did not significantly affect the overall efficiency of viral DNA replication. These results indicate a role of PML and PML-NBs in post-replication DNA processing, and suggest that PML-NBs become linked to sites of viral DNA synthesis due to a role of these structures in DNA metabolism.