A novel role of Dnl4/Lif1 in non-homologous end joining repair complex assembly

Abstract

DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage. The DNA damage response initiated by DSBs includes cell cycle arrest. Misrepair of DSBs has been linked with cancer, immunodeficiency, and neurodegeneration in humans. Two major pathways have evolved to repair these cytotoxic events: (1) homologous recombination (HR), in which a homologous sequence, usually the sister chromatid, is used as a template for repair; and (2) non-homologous end joining (NHEJ), in which the broken ends are brought together and repaired in the absence of homology. In humans, NHEJ is the dominant pathway during most phases of the cell cycle. The core factors required to carry out the NHEJ pathway are mostly conserved from yeast to humans, and as such Saccharomyces cerevisiae is an excellent model organism to study NHEJ proteins and the mechanism by which they repair DSBs. In S. cerevisiae, seven core proteins assemble into three complexes that are genetically required for NHEJ---yKu70 and yKu80 (yKu); Mre11, Rad50, and Xrs2 (MRX); and Dnl4, Lif1 (Dnl4/Lif1). In addition, the haploid-specific Nej1 protein, the deletion of which results in NHEJ defects similar to those caused by genetic inactivation of yKu or Dnl4, has been shown to interact with Dnl4/Lif1. However the exact function of Nej1 has not yet been elucidated. In this study, we examined the temporal order of assembly of core components of the NHEJ pathway required for the formation of a macro-molecular NHEJ repair machine both by in vitro biochemical and in vivo chromatin immunoprecipitation assays. Our findings demonstrated that yKu is the first protein complex to assembly at a DSB and subsequently recruits Dnl4/Lif1. Notably, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs but not on in vitro DNA substrates designed to mimic double strand breaks. yKu and Dnl4/Lif1 proceed to recruit Mre11/Rad50/Xrs2, the end bridging factor, into the nucleoprotein NHEJ complex. In addition, yKu and Dnl4/Lif1 attenuate homologous recombination by inhibiting DNA end resection. We have uncovered a novel and key role of Dnl4/Lif1 in determining DSB repair pathway choice by participating at an early stage of DSB engagement to recruit subsequent proteins in addition to providing the DNA ligase activity that completes NHEJ.