Mycobacteriophage Exploit NHEJ to Facilitate Genome Circularization Robert S. Pitcher, Louise M. Tonkin, James M. Daley, Phillip L. Palmbos, Andrew J. Green, Tricia L. Velting, Anna Brzostek, Malgorzata Korycka-Machala, Steve Cresawn, Jaroslaw Dziadek, Graham F. Hatfull, Thomas E. Wilson, Aidan J. Doherty  Molecular Cell  Volume 23, Issue 5, Pages 743-748 (September 2006) DOI: 10.1016/j.molcel.2006.07.009 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Ω-Ku and Cd-Ku Bind dsDNA Electrophoretic-mobility shift assays were employed to characterize the ability of (A) Ω-Ku and (B) Cd-Ku to bind DNA. Reaction mixtures contained 10 nM labeled DNA duplex (with blunt-ended or with 3′ or 5′ overhangs as illustrated). Lane 1, labeled DNA only; and lanes 2–6, increasing amounts of protein added to DNA (0.05, 0.125, 0.25, 0.5, and 0.75 μM). (C) Ω- and Cd-Ku also super shifted linearized pUC18 plasmid DNA separated on agarose gels. Reaction mixtures contained dsDNA (digested with EcoRI) and (0–15 μM) protein. Lane 1, DNA alone; and lanes 2–6, increasing amounts of protein added to DNA (1, 2, 5, 10, and 15 μM). Molecular Cell 2006 23, 743-748DOI: (10.1016/j.molcel.2006.07.009) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Ω-Ku and Cd-Ku Recruit and Stimulate Ms-LigD (A) Complementary end joining by Ms-LigD in the presence of Ω- and Cd-Ku. Lane 1, 60 nM Ω- or Cd-Ku; and lanes 2–7, 20 nM Ms-LigD with 0, 10, 20, 40, 80, and 125 nM Ω- or Cd-Ku; and lane 8, 20 nM Ms-LigD and 20 nM Ms-Ku. (B) Ms-LigD and Ms-PolDom super shifted both the Ω- and Cd-Ku's and the Ω-Ku, respectively. Reaction mixtures contained 10 nM labeled dsDNA 33 bp duplex. Lane 1, labeled probe only; lane 2, 0.8 μM Ω-Ku; lane 3, 0.8 μM Cd-Ku; lane 4, 0.5 μM Ms-PolDom; lanes 5–7, 0.2, 0.4, and 0.8 μM Ω-Ku in addition to Ms-PolDom (0.5 μM); lane 8, 0.5 μM Ms-LigD; lanes 9–11, 0.2, 0.4, and 0.8 μM Ω-Ku in addition to Ms-LigD (0.5 μM); and lanes 12–14, 0.2, 0.4, and 0.8 μM Cd-Ku in addition to Ms-LigD (0.5 μM). Molecular Cell 2006 23, 743-748DOI: (10.1016/j.molcel.2006.07.009) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Ω-Ku Cooperates with Mycobacterial Ligase D to Reconstitute NHEJ in Yeast Bacterial and phage Ku and LigD proteins were expressed in dnl4 yku70 yeast strains lacking endogenous NHEJ in the indicated combinations, and NHEJ efficiency was examined by plasmid recircularization and chromosomal suicide deletion assays as described in the Experimental Procedures. (A) Plasmid DSBs were created with either NsiI (4 base 3′ overhangs, top) or NcoI (4 base 5′ overhangs, bottom). NHEJ capacity is expressed as the Met+ transformation efficiency by these plasmids normalized to the His+ transformation efficiency by a cotransformed circular HIS3-marked plasmid. (B) Chromosomal DSBs in ADE2 were created by expressing the mega-endonuclease I-SceI (4 base 3′ overhangs). NHEJ capacity is expressed as the fraction of cells that survived I-SceI induction as Ade+ (∼1% for wild-type yeast). Results are plotted on a log scale. Each bar represents the mean ± standard deviation (error bar) from three independent experiments. For each assay, Ω-Ku stimulated LigD-dependent NHEJ, although to different extents in plasmid and chromosomal assays as compared to Mt-Ku and Ms-Ku. Molecular Cell 2006 23, 743-748DOI: (10.1016/j.molcel.2006.07.009) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Ms-LigD, but Not Ms-Ku, Is Required for Omega and Corndog Plaque Formation in M. smegmatis (A) Plaques formed when either wild-type or ΔKu M. smegmatis strains were infected with Omega or Corndog phage lysate. However, no plaques formed when either ΔLigD or ΔLigD, ΔKu strains were infected with Omega or Corndog phage lysate. (B) The DNA ligase, but not the polymerase, activity of Ms-LigD was required for plaque formation. Plaques appeared when the LigD deletion strain is complemented with Ms-LigD expressed from an acetamide-inducible plasmid. Plaques also formed when the LigD deletion strain was complemented with Ms-LigD (containing catalytic mutants that prevent polymerase activity) were infected with either phage lysate. However, plaques did not appear when a LigD deletion strain complemented with Ms-LigD (containing a catalytic mutation [K484A] that prevented ligase activity) was infected with either phage lysate. Molecular Cell 2006 23, 743-748DOI: (10.1016/j.molcel.2006.07.009) Copyright © 2006 Elsevier Inc. Terms and Conditions