DNA looping by Ku and the DNA-dependent protein kinase - PubMed (original) (raw)
Comparative Study
DNA looping by Ku and the DNA-dependent protein kinase
R B Cary et al. Proc Natl Acad Sci U S A. 1997.
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 Ku70/86 heterodimer and an approximately 460-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 protein-nucleic 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.
Figures
Figure 1
SDS/PAGE analysis of HeLa DNA-PK and recombinant Ku70/86. (A) 100 ng of Ku70/86 (lane b) and 100 ng of DNA-PKcs (lane c) purified from HeLa cell nuclear extract was resolved by 10% SDS/PAGE and analyzed by silver staining. Molecular mass markers (lane a) are as follows: Myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97.4 kDa), serum albumin (66.2 kDa), and ovalbumin (45.0 kDa). (B) Purified recombinant Ku70/86 (5 μg) (lane b) was resolved by 10% SDS/PAGE and analyzed by Coomassie blue staining. Molecular mass markers (lane a) are as follows: Myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45.0 kDa), and carbonic anhydrase (31.0 kDa).
Figure 2
AFM and electron microscopy of DNA-PK complexes. (A) Reactions containing DNA-PKcs and a linear 660-bp DNA fragment were examined by AFM. Association between DNA-PKcs and DNA (arrow) was observed but uncommon compared with reactions that also contained Ku. (B) In the presence of recombinant Ku, DNA-PKcs was frequently seen associated with the DNA fragment (white arrows). Black arrows indicate unbound DNA-PKcs. Samples in A and B were mounted and imaged without fixation. (C) Linearized pUC19 incubated in the presence of Ku and DNA-PKcs. Size differences allowed the DNA-PKcs (black arrowhead) to be distinguished from recombinant Ku particles (white arrowhead). End-bound (long arrow) and internally bound DNA-PK (short arrow) were both commonly observed. (D and E) Electron microscopy of rotary-shadowed DNA-PK/DNA complexes assembled using HeLa-purified Ku revealed similar DNA-PK behavior. (D) A DNA-PK complex bound internally to linearized pUC19. The arrow indicates the DNA end. (E) An example of an end-bound DNA-PK complex. Bar in A is 100 nm for A_–_C. Bar in D is 100 nm for D and E.
Figure 3
Qualitative examination of linearized pUC19 incubated with either recombinant Ku (A and B) or HeLa Ku (C and D) revealed the formation of Ku-associated loops. Clusters of Ku particles were common (arrows in B and D). For statistical analysis, a 660-bp DNA fragment was used to quantitate the looping effect initially observed for Ku and DNA-PK on linearized pUC19. For these studies, all samples were mounted and imaged without fixation. (E_–_H) Examples of looped 660-bp DNA formed in the presence of recombinant Ku. (I) Multi-DNA complexes were also observed at an increased frequency in the presence of recombinant Ku. (J_–_M) Loops formed in the presence of Ku and the DNA-PKcs. A free DNA-PKcs particle is visible in the lower right corner of J. Bar in M is 100 nm for A_–_C and E_–_M. Bar in D is 100 nm for D.
Figure 4
DNA-binding and gel filtration analysis of recombinant Ku70/86. (A) Electrophoretic mobility shift analysis of recombinant Ku70/86. DNA (10 pmol) was incubated with the indicated amounts of Ku70/86 and analyzed by gel electrophoresis as described. Results were obtained by autoradiography of the dried gel. (B) Samples from fractions 59–72 of Superdex 200 column chromatography runs of Ku70/86 alone or Ku70/86 in the presence of a 24-bp double-stranded DNA fragment were resolved by 10% SDS/PAGE and analyzed by silver staining. Molecular mass markers (lane M) are as follows: Myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45.0 kDa), and carbonic anhydrase (31.0 kDa).
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