Autonomous DNA replication in human cells is affected by the size and the source of the DNA.

Abstract

We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.In this study we investigated the genetic requirements for origin of replication function in human cells. The study utilizes a system we have developed that allows us to test fragments of DNA for autonomous replication when introduced into human tissue culture cells in both long-and short-term assays. This system should ultimately permit us to deduce the DNA sequence requirements for autonomous replication. It is likely that this information will be relevant for chromosomal replication as well.Over the course of many cell divisions, extrachromosomal vectors are lost from the nuclei of mammalian cells unless they have a means of nuclear retention. The long-term replication assay that we developed overcomes the problem of plasmid loss by using a replication-defective vector derived from Epstein-Barr virus (EBV) that permits linked sequences to be retained in human cells (22). When random human DNA was cloned into this vector and introduced into human cells, we found that a large number of fragments could confer replication ability on the vector in a long-term assay. The resulting plasmids were stably maintained for at least 2 months in human cells.The fragments could also provide for replication in our short-term assay (22). In this assay, the fragments are moved to a prokaryotic vector that lacks all viral sequences, and DNA is harvested 4 days after transfection, since the plasmids have no means of nuclear retention. These experiments indicated that the human DNA fragments that we had isolated contained sequences involved in the initiation of DNA replication in human cells. Subsequent studies have shown that, like the chromosomes, these plasmids undergo replication once per cell cycle in human cells (unpublished data). This characteristic makes the autonomously replicating system a reasonable model for chromosomal replication.The hu...

Autonomous replication of human chromosomal DNA fragments in human cells.

“…It has been reported that almost any human genomic DNA fragments longer than a certain length replicate autonomously in human cells and that the efficiency of replication varies depending on the size of the fragment (Heinzel et al, 1991). Our results also showed that not only HER segments but segments excluded during HER selection are able to replicate in human cells.…”

LCR-mediated, long-term tissue-specific gene expression within replicating episomal plasmid and cosmid vectors

“…However, our findings are consistent with previous observations that found plasmid EBV-based vectors containing randomly selected large (у16 kb) human genomic DNA fragments, conferred efficient episomal replication in mammalian cells. 11,40 This may involve the presence of either additional genomic origins of replication, 11,12,40 or S/MAR elements 39 or both. While the retention of episomes within cells is clearly important, our work indicates that the maintenance of tissue-specific transgene expression from within REVs is also a crucial consideration.…”

Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain

“…However, replication efficiency varies with the size of the inserted fragment (33), suggesting either that the density of replicators in mammalian genomes is very high, or that sequence requirements for origin function in this assay are minimal, but larger fragments are more efficiently retained.…”