The leucine rich region of DNA-PKcs contributes to its innate DNA affinity.
DNA-PK is a protein complex that consists of a DNA-binding, regulatory subunit [Ku] and a larger approximately 465 kDa catalytic subunit [DNA-PKcs], a serine/threonine protein kinase. The kinase activity of DNA-PKcs resides between residues 3745 and 4013, a PI3 kinase domain. Another recognized domain within this large protein is a leucine zipper (LZ) motif or perhaps more appropriately designated a leucine rich region (LRR) that spans residues 1503-1602. Whereas, DNA-PK's kinase activity has been shown to be absolutely indispensable for its function in non-homologous end joining (NHEJ), little is known about the functional relevance of the LRR. Here we show that DNA-PKcs with point mutations in the LRR can only partially reverse the radiosensitive phenotype and V(D)J recombination deficits of DNA-PKcs deficient cells. Disruption of the LRR motif affects the ability to purify DNA-PKcs via its binding to DNA-cellulose, but does not affect its interaction with Ku or its catalytic activity. These data suggest that the LRR region of DNA-PKcs may contribute to its intrinsic DNA affinity, and moreover, that intrinsic DNA binding is important for optimal function of DNA-PKcs in repairing double strand breaks in living cells.
Gupta
Shikha
S
Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.
Meek
Katheryn
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D056970:Co-Repressor Proteins; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015321:Gene Rearrangement; D006801:Humans; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D015530:Nuclear Matrix / Q000201:enzymology; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011836:Radiation Tolerance; D012097:Repressor Proteins / Q000378:metabolism
C117400:C1D protein, human; D056970:Co-Repressor Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human