Opposite replication polarity of the germ line c-myc gene in HeLa cells compared with that of two Burkitt lymphoma cell lines.

Abstract

To study the cell type specificity of the direction of replication of the human c-myc genes and the relationship of replication polarity to transcriptional activity, we analyzed the directions of replication of the c-myc genes in two Burkitt lymphoma cell lines, CA46 and ST486, and in HeLa cells. On the basis of in vitro runoff replication of forks initiated in intact cells, we found that transcribed c-myc genes in the germ line configuration in HeLa cells were replicated in the direction of transcription from origins in the 5'-flanking DNA, while the repressed, unrearranged c-myc genes of CA46 and ST486 cells were replicated in the antitranscriptional direction. In contrast, the transcribed c-myc genes of CA46 cells were replicated in the transcriptional direction, while the translocated, amplified c-myc genes of ST486 cells showed no preferred polarity of replication. The data also provided evidence for the existence of an endogenous barrier to DNA polymerases in the flanking DNA immediately 5' to the HeLa c-myc genes.The replication of eucaryotic chromosomes is controlled through the selection of sites for the initiation of DNA synthesis (2,6,7,10,12,14,19,21,24,32,39) and through the temporal programming of the activity at these sites (10,12,19,23). Combined with evidence for the existence of proteins which recognize the replication origins of viral minichromosomes (11,17,28,44), these results suggest that chromosome replication can be regulated in trans by DNA sequence-specific protein binding.That the activity of eucaryotic genes is related to the replication of chromatin is supported by several observations that transcribed genes are replicated early in the S phase of the cell cycle (8,9,16,18) and by the demonstration that the efficient transcription of certain transfected genes requires the replication of their chromatin template (13, 42). Moreover, Miller and Nasmyth (31) have demonstrated that passage through the S phase is necessary for repression of the silent mating type loci in Saccharomyces cerevisiae. Smithies (37) has proposed that the processes of replication and transcription are linked such that transcriptionally active genes are replicated from upstream origins, whereas inactive genes are replicated from either upstream or downstream origins. Consistent with this proposal, we have shown that the avian histone H5 genes are replicated in the transcriptional direction in embryonic erythrocytes, where they are expressed, but in the antitranscriptional direction in lymphoblastoid cells and chicken embryo fibroblasts, where they are quiescent (43). In comparison, the avian alpha-pi and alpha-D globin genes are replicated in the transcriptional direction both in cells in which they are active (erythrocytes) and in cells in which they are inactive (lymphoblastoid cells and fibroblasts) (26; Y. I. Lindstrom and M. Leffak, manuscript in preparation).To investigate further the relationship between replication polarity and transcriptional activity, we used the in vitro runoff replication (IVR) assay (2...

A DNA replication-arrest site RTS1 regulates imprinting by determining the direction of replication at mat1 in S. pombe

“…For example, arrest sites may act by regulating recombination or by promoting differential chromatin assembly of sister chromatids at key developmental loci during replication (Seidman et al 1979;Shinahara and Stillman 1999). In support of this possibility, differences in the direction of replication have been observed between expressed and unexpressed genes in human cell lines (Trempe et al 1988;Leffak and James 1989). Whether replication arrest sites are involved in regulation at these genetic loci remains to be explored.…”

A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus.

“…In large part this is due to the fact that prokaryotic, viral, and yeast origins support autonomous replication of episomal DNAs, whereas mammalian origins usually do not (14). As an alternative, several sensitive assays for chromosomal origin function based on physical and chemical properties of replicating duplex DNAs have been developed (50) and used to identify OBRs which reside near human c-myc (28,48) and aprt (22), the murine adenosine deaminase locus (10,51), and the Chinese hamster dhfr (dihydrofolic acid reductase) (1,5,30) and rhodopsin (20) genes.…”

Genetic Analysis of a Mammalian Chromosomal Origin of Replication

“…Since chromosomal context is an important determinant of origin function in mammalian cells (17,54,56,3,27,33,30), the function of the exogenous ori-ß IR may be sensitive to position effects that were not detected in the uncloned cell pools. If initiation activity of the 5.7 kb fragment is affected by chromosomal context, one might expect to find that initiation activity of ectopic ori-ß would vary among clonal cell lines with different ori-ß integration sites.…”