Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation

Abstract

Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2–3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2–3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation.

Altered Replication in Human Cells Promotes DMPK (CTG)n · (CAG)n Repeat Instability

“…4). The differences in origin activity between the three non-DM1 cell types (HeLa, IMR90, and MM) and the three DM1 cell types (GM04033, GM03987, and 428-12D), therefore, may not be due to (CTG) n · (CAG) n TNR expansion but may be tissue, age, or transformation related, as previously reported (19,24).…”

Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

“…The ability of several versions of the 20mer present within human chromosomes to confer autonomous replication activity when cloned into a plasmid was analyzed by the DpnI resistance assay, which is an indicator of semiconservative DNA replication, as previously described. 12,36,38 HeLa, NSF, WI38, and WI38(SV40) cells (Fig. 1A, B) were transfected with one of the following constructs (Suppl.…”

“…30 Several studies have shown that increased origin usage is a common feature in transformed compared with normal cells. [31][32][33][34][35][36][37][38][39][40] Considering the importance of chromatin structure in origin activation, we examined the epigenetic environment at replication origins as well as their association with chromatin modifying factors in both transformed and normal cells. A comparative analysis of the chromatin structure encompassing replication origins in normal and transformed cells will aid in the understanding of why some origins are more active in transformed compared with normal cells, [33][34][35][36]38 having an elevated association of pre-RC proteins, 40 and thus may give us insight into the mechanisms that regulate the initiation of DNA replication in normal cells and how it may become deregulated by cellular transformation.…”

Forced binding of the origin of replication complex to chromosomal sites inDrosophilaS2 cells creates an origin of replication

“…Differences in replication initiation between transformed and non-transformed cells have already been documented (e.g. Dorn et al, 2009;Di Paola et al, 2010).…”