In vitro rejoining of DNA double strand breaks: a comparison of genomic-DNA with plasmid-DNA-based assays

Abstract

Purpose: To evaluate potential similarities between the enzymatic activities required to rejoin DNA double strand breaks (dsb) in an in vitro assay based on genomic DNA and an in vitro assay based on plasmid DNA. Because the latter assay is simpler and faster, it should be preferred for the characterization of repair factors if both assays are found to probe for the same activities. If, however, the enzymatic requirements for dsb rejoining are different between the two assays, both should be used as they are likely to play complementary roles in the characterization of repair factors. Materials and methods : A cell-free assay has been used, developed to study rejoining DNA dsb induced by radiation in 'naked' DNA prepared from agarose embedded cells using an extract of HeLa cells as a source of enzymes. Also employed was an in vitro assay using the ligation of linearized plasmid DNA to model dsb rejoining. Results : Evidence is presented that, under the conditions employed, different sets of activities are involved in the ligation of linearized plasmid DNA and in the rejoining of dsb in 'naked' genomic DNA. Optimal rejoining of dsb induced in genomic DNA is observed with cytoplasmic cell extract at 37 C, whereas optimal ligation of plasmid DNA is observed with nuclear extract at 25 C. Rejoining of dsb in genomic DNA comes to a near halt at 14 C, but plasmid DNA ligation proceeds at significant rates at this temperature. Furthermore, the activities required for the rejoining of dsb induced in genomic DNA are partly stable to heating at 50 C for 1 h, whereas activities required for the ligation of plasmid DNA are completely inactivated by a similar treatment. Both reactions require ATP for optimal performance, and in both, DNA joining is inhibited at high ATP concentrations. Conclusions : These observations indicate that the plasmid-and the genomic-DNA-based assays probe for, at least partly, different sets of activities and therefore are expected to play complementary roles in the purification and characterization of activities involved in dsb rejoining.