DNA ligase I is an in vivo substrate of DNA-dependent protein kinase and is activated by phosphorylation in response to DNA double-strand breaks.
DNA-dependent protein kinase (DNA-PK) phosphorylates several cellular proteins in vitro, but its cellular function and natural substrate(s) in vivo are not established. We reported activation of DNA ligase in cultured normal human epidermal keratinocytes (NHEK) on exposure to the DNA-damaging compound bis-(2-chloroethyl) sulfide. The activated enzyme was identified as DNA ligase I, and this activation was attributed to phosphorylation of the enzyme. Here, we show that the phosphorylation is mediated by DNA-PK and that DNA ligase I is one of its natural substrates in vivo. DNA ligase I phosphorylation-cum-activation is a response specific to DNA double-strand breaks. We also demonstrate that affinity-purified inactive DNA ligase I is phosphorylated and activated in vitro by HeLa Cell DNA-PK confirming the in vivo observations. The findings specify the roles of DNA-PK and DNA ligase I in mammalian DNA double-strand break repair.
Bhat
K Ramachandra
KR
Department of Chemistry, Lincoln University, Pennsylvania 19352-0999, USA.
Benton
Betty J
BJ
Ray
Radharaman
R
D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009151:Mustard Gas / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D013379:Substrate Specificity
C000607892:LIG1 protein, human; D004247:DNA; D051747:DNA-Activated Protein Kinase; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D009151:Mustard Gas