Biochemical evidence for Ku-independent backup pathways of NHEJ
Authors: Ange Ronel Perrault
Wei Qin
Yoshihiko Takeda
George Iliakis
Hongyan Wang
Huichen Wang
Journal: Nucleic acids research (2003)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: DSBs; NHEJ; DNA-PKcs; Ku; DNA ligase IV
Affiliations: Department of Radiation Oncology, Division of Experimental Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA.

Abstract:  Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a non-homologous end joining (NHEJ) apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4 and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with an order of magnitude slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group and frequently joins incorrect ends. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. The present study investigates the role of Ku in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient, error-free, end joining observed in such in vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite the fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA end joining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing end joining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts in line with the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the affinity of Ku for DNA ends, particularly when cooperating with DNA-PKcs, suppresses B-NHEJ by quickly and efficiently binding DNA ends and directing them to D-NHEJ for rapid joining. A chromatin-based model of DNA DSB rejoining accommodating biochemical and genetic results is presented and deviations between in vitro and in vivo results discussed. 

Retracted: No

PubPeer comments:
 Figure 2B. Much more similar than you would expect. https: imgur.com ax2SmAk , 
 Figure 3B. Much more similar than you would expect. See: https: imgur.com ZsZbpHd , 
 2020 Expression of Concern. https: academic.oup.com nar advance-article doi 10.1093 nar gkaa228 5815011 The Authors and Editors wish to jointly publish an Expression of Concern regarding the above article. The Editors wish to alert the readers that questions have been raised about the validity of some of the figures presented in this article. In Figure 2B, some lanes have been duplicated as described below: The sample of HeLa without NHS was lost, the authors duplicated the lane of HeLa with 0.05u03bcl NHS. The sample of M059-J with 0.2u03bcl of NHS was lost, the authors duplicated the lane of M059-J with 0.025u03bcl of NHS. Lane 7 of Figure 3B (M059-J, probe only) has been copied from a different experiment. While these irregularities may not fundamentally affect the results and conclusions of the article, the Editors nonetheless note that such manipulations of data and corresponding figures is unacceptable. The authors concur and deeply regret the transgression. Keith Fox, Senior Executive Editor, Nucleic Acids Research Barry Stoddard, Senior Executive Editor, Nucleic Acids Research ,