DNA replication, cell cycle progression and the targeted gene repair reaction

Abstract

Single-stranded oligonucleotides (ssODNs) can direct base changes in mammalian cells and influence changes in phenotype. The mechanism by which ssODNs alters the sequence is being revealed by studies carried out in model systems. In the long run, this information will provide the basis for clinical protocols designed to target genetic diseases. It is now clear that DNA replication plays an important part in the gene repair reaction. Here, we examine gene repair as a function of the amount of cells passing through S phase. We find that cells in mid to late S are most amenable to gene repair, and reaction manipulations that enrich the population of cells in S phase naturally lead to elevated correction frequencies. Our data suggest that these intra-S sub phases support higher levels of repair independent of transfection efficiencies or the rates of replication. A preliminary gene expression profile of cells in the most amenable correction phase indicates that the levels of cyclin G(2), cyclin H, CDK12A and CDK12B are raised significantly. Taken together, our data identify sections of S phase that enable higher levels of gene repair and establish a mechanistic framework for the use of gene repair in clinical setup.