Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex

J Biol Chem. 2016 Oct 14;291(42):21893-21902. doi: 10.1074/jbc.M116.746198. Epub 2016 Aug 29.

Abstract

The synthesis, processing, and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and coordinates the activity of several DNA replication proteins, including the enzymes flap endonuclease 1 (FEN-1) and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. Although it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Because DNA ligase I has been reported to be ubiquitylated, we used a proteomic approach to map ubiquitylation sites and screen for DNA ligase I-associated E3 ubiquitin ligases. We identified three ubiquitylated lysine residues and showed that DNA ligase I interacts with and is targeted for ubiquitylation by DCAF7, a specificity factor for the Cul4-DDB1 complex. Notably, knockdown of DCAF7 reduced the degradation of DNA ligase I in response to inhibition of proliferation and replacement of ubiquitylated lysine residues reduced the in vitro ubiquitylation of DNA ligase I by Cul4-DDB1 and DCAF7. In contrast, a different E3 ubiquitin ligase regulates FEN-1 turnover. Thus, although the expression of many of the genes encoding DNA replication proteins is coordinately regulated, our studies reveal that different mechanisms are involved in the turnover of these proteins.

Keywords: DNA ligase I; DNA replication; cell cycle; proliferating cell nuclear antigen (PCNA); proteasome; protein turnover; proteolysis; ubiquitin; ubiquitin ligase; ubiquitylation.

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Cell Line
  • Cell Proliferation / physiology
  • Cullin Proteins / genetics
  • Cullin Proteins / metabolism*
  • DNA Ligase ATP / genetics
  • DNA Ligase ATP / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Female
  • Flap Endonucleases / genetics
  • Flap Endonucleases / metabolism
  • Gene Expression Regulation / physiology
  • Humans
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / metabolism*
  • Proteolysis*
  • Ubiquitination / physiology

Substances

  • Adaptor Proteins, Signal Transducing
  • Cullin Proteins
  • DCAF7 protein, human
  • DDB1 protein, human
  • DNA-Binding Proteins
  • Multienzyme Complexes
  • Flap Endonucleases
  • FEN1 protein, human
  • DNA Ligase ATP