Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA ligase IV (Lig4), XRCC4, and DNA-PK are key mammalian proteins for DNA repair and V(D)J recombination via nonhomologous end joining.
• Previous studies indicated Lig4 as an in vitro substrate for DNA-PK, but its in vivo phosphorylation status remained unknown.

Purpose of the Study:

• To investigate the in vivo phosphorylation of Lig4 by DNA-PK.
• To identify DNA-PK phosphorylation sites on Lig4.
• To determine the functional consequences of Lig4 phosphorylation on its DNA repair activity and stability.

Main Methods:

• Expression of full-length Lig4 construct in cells.
• In vitro kinase assays using Lig4-XRCC4 complex and DNA-PK.
• Tandem mass spectrometry for phosphosite identification.
• Site-directed mutagenesis to create alanine substitution and phosphomimetic mutants.
• Assessment of Lig4 protein stability and DNA end joining activity.

Main Results:

• Full-length Lig4 was expressed as a phosphoprotein in cells.
• The Lig4-XRCC4 complex was an in vitro substrate for DNA-PK.
• DNA-PK phosphorylation sites were identified at Thr-650 and potentially Ser-668/Ser-672 in human Lig4.
• Lig4 phosphorylation was not essential for its DNA end joining activity.
• Mutations at phosphorylation sites affected Lig4 protein stability, with a phosphomimetic mutation restoring wild-type stability.
• DNA-PK negatively regulated Lig4 protein stability.

Conclusions:

• Lig4 is phosphorylated in vivo by DNA-PK, with specific sites identified.
• Lig4 stability is regulated by interactions with XRCC4, phosphorylation status, and potentially protein conformation.
• DNA-PK plays a role in modulating Lig4 stability, impacting DNA repair pathways.