Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg2+. At 0.5 mM Mg2+, where only DNA ligase IV is expected to retain activity, low levels of end joining (∼10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg2+ in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B-NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observations to studies of NHEJ in vivo and in vitro are discussed.

Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein

We attempted to devise a transcription system in which a particular DNA sequence of interest could be inducibly expressed under the control of a modified polymerase III (pol III) promoter. Its activation requires a mutated transcription factor not contained endogenously in human cells. We constructed such a promoter by fusing elements of the β-lactamase gene of Escherichia coli, containing a modified TATA-box and a pol III terminator, to the initiation region of the human U6 gene. This construct functionally resembles a 5′-regulated pol III gene and its transcribed segment can be exchanged for an arbitrary sequence. Its transcription in vitro by pol III requires the same factors as the U6 gene with the major exception that the modified TATA-box of this construct only interacts with a TATA-binding protein (TBP) mutant (TBP-DR2) but not with TBP wild-type (TBPwt). Its transcription therefore requires TBP-DR2 exclusively instead of TBPwt. In order to render the system inducible, we fused the gene coding for TBP-DR2 to a tetracycline control element and stably transfected this new construct into HeLa cells. Induction of such a stable and viable clone with tetracycline resulted in the expression of functional TBP-DR2. This system may conceptually be used in the future to inducibly express an arbitrary DNA sequence in  vivo under the control of the above mentioned promoter.