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^,

Abstract

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, induding suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Maryland 21010-5400, and Department of Chemistry, Lincoln University, Pennsylvania 19352-0999 Received July 28, 2005; Revised Manuscript Received January 9, 2006 ABSTRACT: 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.

RETRACTED: Sealing of Chromosomal DNA Nicks during Nucleotide Excision Repair Requires XRCC1 and DNA Ligase IIIα in a Cell-Cycle-Specific Manner

“…In mitotic cells, Lig1 is hyperphosphorylated (at serines Ser51, Ser66, Ser76, and Ser91) and functionally inactive (Rossi et al, 1999;Ferrari et al, 2003). Although Lig1 is present in quiescent human keratinocytes, the protein is inactive and requires phosphorylation for its activation (Bhat et al, 2006). In agreement with these data, we identified hyperphosphorylated Lig1 in G2/M and hypophosphorylated Lig1 in both UV-or mock-treated quiescent cells (G0) cells.…”

A Large-Scale Quantitative Proteomic Approach To Identifying Sulfur Mustard-Induced Protein Phosphorylation Cascades

“…Earlier work by our group also detected increased phosphorylation levels of this protein following SM exposure in HaCaT cells . LIG1 (IPI00219841) was previously reported to be activated via phosphorylation in response to SM-induced DNA damage . Consistent with these results, a phosphopeptide from LIG1 was detected greater than 2-fold higher in SM-treated cells.…”

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

“…The DNA-PK-mediated phosphorylation of LigI activates the enzyme and is observed both in vitro and in vivo [130]. The observed in vivo phosphorylation of LigI corresponds with the generation of double-stranded DNA breaks and may be specific for the role of LigI in non-homologous end joining.…”