DNA looping by Ku and the DNA-dependent protein kinase

Abstract

The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70͞86 heterodimer and an Ϸ460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and proteinnucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.

Regulation and mechanisms of mammalian double-strand break repair

“…KU, DNA-PKcs, and XRCC4 all promote end-toend association of linear DNAs, both alone and in concert with each other (Cary et al, 1997;Pang et al, 1997;Yaneva et al, 1997;DeFazio et al, 2002). Such an association involves interaction between two molecules of KU (Cary et al, 1997) or DNA-PKcs (DeFazio et al, 2002), one on each DNA end.…”

“…Although KU tends to cling to the DNA end, it can translocate into the interior, allowing multiple KU heterodimers to load onto longer DNA molecules (Mimori and Hardin, 1986;Paillard and Strauss, 1991;Cary et al, 1997;Pang et al, 1997;Yaneva et al, 1997). The end-bound KU occupies approximately 16-18 bp, as indicated by X-ray crystallography , DNase footprinting and ultraviolet crosslinking (Yoo and Dynan, 1999).…”

“…A diverse body of evidence from studies with cell-free extracts and purified proteins implicates both KU and XRCC4/ligase IV in the alignment of partially cohesive ends for gap filling (Cary et al, 1997;Pang et al, 1997;Yaneva et al, 1997;Chen et al, 2000;Feldmann et al, 2000;Chen S et al, 2001;Lee et al, 2003). The gapfilling polymerase is most likely either DNA polymerase m (POLm or POL l).…”

“…Such an association involves interaction between two molecules of KU (Cary et al, 1997) or DNA-PKcs (DeFazio et al, 2002), one on each DNA end. The effects of KU and DNA-PK on ligation of compatible ends by XRCC4/ ligase IV are complex, under some conditions promoting ligation as much as 20-fold (Nick McElhinny et al, 2000), while under other conditions inhibiting ligation or favoring inter-over intra-molecular ligation .…”

Binding of the DNA-dependent protein kinase catalytic subunit to Holliday junctions

“…Fourthly, we demonstrated that the cross-linked DNA-PK CS subunit retains its enzymic activity and is capable of phosphorylating itself as well as heterologous protein acceptors (Figure 3). Finally, the identification of DNA-PK CS bound to interior positions of DNA duplexes is not without precedent, as previous atomic force microscopy studies have shown trapping of the enzymic subunits both at the DNA ends and internally [37]. We noted, however, that this binding to internal positions of linear substrates is suppressed in the context of the nuclear protein environment ( Figure 7B).…”