FIGURE 6.

The de novo dTMP synthesis pathway is associated with episomal DNA in tandem chromatin immunoprecipitations. HeLa cells were transfected with pBABE-Puro-SV40 LT vector and selected for using puromycin. Clones were isolated and immunoblots performed against SV40 LT to ensure that clones were expressing SV40 LT (D). Tandem ChIP assays were performed as described under “Materials and Methods” using antibodies directed against PCNA and then antibodies directed against non-immune IgG, SHMT1, TYMS, or DHFR. Three separate SV40 LT-expressing clones were subjected to tandem ChIP in duplicate. Error is expressed as S.E. (error bars). Regions of the pBABE-Puro-SV40 LT vector were probed for the presence of SHMT1, TYMS, or DHFR using real-time PCR (A–C). All three enzymes precipitated regions of the vector, including the large T antigen coding region (A), SV40 origin of replication (B), and downstream region of the SV40 origin (C). The highest occupancy was observed around the SV40 origin.