Analysis of DNA-dependent protein kinase-mediated DNA end joining by two-photon fluorescence cross-correlation spectroscopy.
Nonhomologous end joining (NHEJ) is the primary mechanism by which mammalian cells repair DNA double-strand breaks (DSBs). Proteins known to play a role in NHEJ include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the Ku 70/Ku 80 heterodimer (Ku), XRCC4, and DNA ligase IV. One of the main roles of the DNA-PKcs-Ku complex is to bring the ends of the DSB together in a process termed synapsis, prior to end joining. Synapsis results in the autophosphorylation of DNA-PKcs, which is required to make the DNA ends available for ligation. Here, we describe a novel assay using two-photon fluorescence cross-correlation spectroscopy that allows for the analysis of DNA synapsis and end joining in solution using purified proteins. We demonstrate that although autophosphorylation-defective DNA-PKcs does not support DNA ligase-mediated DNA end joining, like wild-type (WT) DNA-PKcs, it is capable of Ku-dependent DNA synapsis in solution. Moreover, we show that, in the presence of Ku, both WT DNA-PKcs and autophosphorylation-defective DNA-PKcs promote the formation of multiple, large multi-DNA complexes in solution, suggesting that, rather than align two opposing DNA ends, multiple DNA-PK molecules may serve to bring multiple DNA ends into the NHEJ complex.
Department of Chemistry, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, T2N 1N4, Canada.
Dennis
D
Merkle
Wesley D
WD
Block
Yaping
Y
Yu
Susan P
SP
Lees-Miller
David T
DT
Cramb
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D006801:Humans; D013050:Spectrometry, Fluorescence / Q000379:methods
D004247:DNA; D051747:DNA-Activated Protein Kinase; D011088:DNA Ligases