Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding

DNA Repair (Amst). 2014 Dec;24:37-45. doi: 10.1016/j.dnarep.2014.10.003.

Abstract

LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure–function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4–Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair
  • DNA Ligase ATP
  • DNA Ligases / chemistry
  • DNA Ligases / genetics*
  • DNA Ligases / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Mutation
  • Protein Stability
  • Protein Structure, Tertiary*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • DNA-Binding Proteins
  • DNL4 protein, S cerevisiae
  • LIF1 protein, S cerevisiae
  • LIG4 protein, human
  • Saccharomyces cerevisiae Proteins
  • XRCC4 protein, human
  • DNA Ligases
  • DNA Ligase ATP