Essential Factors for Incompatible DNA End Joining at Chromosomal DNA Double Strand Breaks In Vivo

Hideaki Ogiwara,Takashi Kohno

doi:10.1371/journal.pone.0028756

Abstract

Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double strand break (DSBs) with incompatible DNA ends, which are often generated by ionizing irradiation. In vitro reconstitution studies have indicated that NHEJ of incompatible DNA ends requires not only the core steps of synapsis and ligation, employing KU80/DNA-PKcs and LIG4, but also additional DNA end processing steps, such as DNA end resection by Artemis and gap-filling by POLλ and POLμ. It seems that DNA end processing steps are important for joining of incompatible DNA ends rather than compatible ends. Despite the fact that DNA end processing is important for incompatible DNA end joining in vitro, the role of DNA processing in NHEJ of incompatible DSBs in vivo has not yet been demonstrated. Here we investigated the in vivo roles of proteins implicated in each step of NHEJ using an assay in which NHEJ of incompatible DNA ends on chromosomal DNA can be assessed in living human cells. siRNA- or inhibitor-mediated impairment of factors in each NHEJ step resulted in a reduction in joining efficiency. Strikingly, stronger effects were observed when DNA end resection and ligation protein functions were impaired. Disruption of synapsis by KU80 and DNA-PKcs impairment, or the disruption of gap filling by POLλ and POLμ depletion, resulted in higher levels of microhomology-mediated joining. The present study indicates that DNA end resection and ligation factors are critical for the efficient joining of incompatible ends in vivo, further emphasizing the importance of synapsis and gap-filling factors in preventing illegitimate joining.

Highlights

Non-homologous end joining (NHEJ) is a system that repairs DNA double strand breaks (DSB) by joining two broken DNA ends without requiring long stretches of homology, while homologous recombination repair (HRR) joins two broken DNA ends by using long (.100 bp) stretches of nucleotide homology [1,2,3]
In NHEJ, a complex consisting of the KU70/KU80 heterodimer and DNA-PKcs mediates the synapsis of two broken DNA ends and is followed by a ligation reaction performed by LIG4/XRCC4/XLF
NHEJ of two broken DNA ends generated by I-SceI endonuclease digestion results in the deletion of the HSV-TK (HSV-thymidine kinase) open reading frame and leads to the production of a transcript that enables the translation of enhanced green fluorescent protein instead of the HSV-TK protein

Summary

Introduction

Non-homologous end joining (NHEJ) is a system that repairs DNA double strand breaks (DSB) by joining two broken DNA ends without requiring long stretches of homology, while homologous recombination repair (HRR) joins two broken DNA ends by using long (.100 bp) stretches of nucleotide homology [1,2,3]. NHEJ of compatible DNA ends requires synapsis and ligation factors in reconstitution systems, in which purified proteins and naked DNAs are reacted in vitro. These synapsis and ligation factors are defined as the core factors of NHEJ [1,3,4,5]. Lack of synapsis factors prompts micro-homology– mediated joining, an alternative mode of NHEJ that does not require synapsis factors [6,7,8,9]

Methods

Results

Conclusion