title: Unraveling the complexities of DNA-dependent protein kinase autophosphorylation.
abstract: DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a "hinge" region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ.
authors.affiliation: College of Veterinary Medicine, Department of Microbiology and Molecular Genetics, and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Neal
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Seiji
authors.initials: S
authors.lastname: Sugiman-Marangos
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Pamela
authors.initials: P
authors.lastname: VanderVere-Carozza
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Mike
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA Departments of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: John
authors.initials: J
authors.lastname: Turchi
authors.affiliation: Departments of Biochemistry and Molecular Biology and Oncology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Susan P
authors.initials: SP
authors.lastname: Lees-Miller
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Murray S
authors.initials: MS
authors.lastname: Junop
authors.affiliation: College of Veterinary Medicine, Department of Microbiology and Molecular Genetics, and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA kmeek@msu.edu.
authors.forename: Katheryn
authors.initials: K
authors.lastname: Meek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002945:Cisplatin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D018736:DNA Adducts / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005799:Genes, Dominant; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D045567:VDJ Recombinases / Q000378:metabolism
chemical_list: D018736:DNA Adducts; D051747:DNA-Activated Protein Kinase; D045567:VDJ Recombinases; D002945:Cisplatin