Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

Xi Chen,Alan E Tomkinson

doi:10.1074/jbc.m110.195024

Abstract

In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway.

Highlights

The double-strand breaks (DSBs) site, whereas the repair of DSBs by nonhomologous end joining (NHEJ) is error-prone
In ChIP assays to measure the steady-state levels of Nej1 at a site-specific DSB generated by HO endonuclease, genetic inactivation of yeast Ku (yKu) markedly reduced the recruitment of Nej1, whereas the absence of either the Lif1 or Dnl4 subunit of the Dnl4-Lif1 complex resulted in a relatively minor increase in Nej1 recruitment (Fig. 1C)
ChIP assays performed without cross-linking were used to demonstrate that the recruitment of Dnl4-Lif1 to an in vivo DSB was dependent upon yKu, Dnl4-Lif1 was critical for the stable retention of yKu at the DSB [19]

Summary

Introduction

The DSB site, whereas the repair of DSBs by nonhomologous end joining (NHEJ) is error-prone. The chip binding surface was regenerated by incubation for 60 s with 0.05% SDS solution, followed by a 30-s incubation with running buffer containing 500 mM NaCl. Deadenylation and Adenylation Assays—To remove the AMP moiety from preadenylated Dnl4, the His-tagged Dnl4Lif1 complex (1 pmol) was incubated with 10 mM pyrophosphate in a final volume of 20 ␮l for 30 min at 25 °C in AMP buffer (60 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM DTT, 50 ␮g/ml BSA, and 150 mM NaCl) to reverse the first step of the ligation reaction in the presence or absence of CBP-Nej1 (2 pmol).

Results

Conclusion