Mycobacterial Ku and Ligase REPORT s Proteins Constitute a Two Component NHEJ Re
ID: 163756 • Letter: M
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Mycobacterial Ku and Ligase REPORT s Proteins Constitute a Two Component NHEJ Repair Machine na Della. Phi Louise M. Tonkin Palmbos, Hui-Mi n Tse Robert S. Pitcher, 1.4 James M. Dale Leana M. Topper. Alan E. Tomkinson Thomas E. Wilson, and, DNA, demonstrating tenmi ig S2 Aidan J. Doherty ig synthesized mammalian cells, repair of DNA brea using DNA nucleotide triphosphates (NTPs) but not and end joining HE) is critical genome DSBs) by nonhomolo. ligation for stability Although the end-brid the steps of NHEJ have been reconstituted in vitro, little is known about end-processing reactions hat occur before ligation. Recen y, functional homologous end bridging and ligation activities have been identified in prokarya Consistent with its homology to polymerases and nuclea es, we demonstrate that DNA lig dependent RNA primase To ex merizat gonucleotides that e D from bacterium tuberculosis (Mt-Lig) possesses a nucleotidyl trans erase generate a nonligatabi riety o ue va- activities, including gap-filling polymerase, t inal transferase, and primase, and is also a 3 to 5' exonuclease activities allo the Mt-Ku and Mt-Lig proteins to table strand Thes e enzyme left). Addition of a p ro, as well as to reconstitute join incompatible DSB ends in that prokaryotic Ku and gase NHEJ in v yeast. These results demonstrate ition, processing, and form a bona fide NHEJ ystem that encodes all the ligation activities required for DSB repair he 5 terminus of the resulted in gap ndica concerted g polymerase and Non hom ogous end-join led to contain a di incompatible DNA en DNA double- ogous to eukaryotic primase (o 12: how- strand breaks (DSBs progressively digested the 3 but not the 3 an cells ever, a requirement primase during duplexes anyotie NHEJ are until reach- bvious. Eukaryotic primases share sig ing the double-strand (ds) region (F Ku80 heterodimer (Ku DNA sequence hon with the Pol X Thus. Mu-Lig possesses 3 2), which have functional ssDNA exo- rans ferase Using DNA substrates that karyotes (3) Ku binds directly to the ucl case acl 13 17 Purified recombinant Mt-Lig generate a 3 -flap adjacent to a of DSBs and has end-bridging activity was an efficient DNA-dependent DNA removed the flap by exonucleolytic digestion as has the Mrell Rad50 Xrs2 (MRX polymerase in template-dependent primer ex- generating a base-paired linear duplex (Fig. complex (6). These factors Lig similarly acted C) At higher concentrations, the nuclease achieve repair pendent RNA polymerase progressed h the microhomology re DSBs generated in vivo have damaged o complementary termini that require processing by nucleases and polymerases to generate Fig. 1. M gatable lemn e molecular mecha multidomain enzyme these reactions are poorly understood polymerase, nu ease, and ligase ac The vcabacterium iuberculosis DNA pair ligase, Mi-Lig (R and L assays with DNA Ku hom plexes that form a non dom gatable nificant homo 25, 0.25 0,5, o M) efficient and possibly nucleases (10-12), suggest he gap process and then jo Right: When a phos phate group was added Cambridge Institute Medical Research, University to the 5 terminus M of Cambridge, Department of Haematology Cambridge CB2 2XY, UK. Department of Pathology, ed intermediate. (B higan Medical School. Ann Arbor, University DNA duplexes wi USA. Molecular Medicine Grad- overhangs were incu uate Program, institute of Biotechnology, The Univer bated wi ncreasing San Antonio, San exas Health Science Center Antonio, TX 78245 3207, USA G Mt-Lig enome Damage and mounts of 0.25, 0.5, and 1 uM Stability Centre. University of Sussex Falmer, Brighton the BN1 9RQ, UK 'Department of Radiation Oncology and on sub Greenebaum Cancer Center, Universi oligome Medicine, Baltimore, MD 21201-15S9, USA. ate and 43 degrada Mt-Lig orme On of Cancer Research products are indicated Beatty Laboratories, London SW3 6lB, UK Chest c) DNA substrates were incubated with increasing amounts of Mt-Lig (0.25, 0.5. and 1 HM). The These authors contributed equally to this wark. the 51-nt oligomer substrate and 48- and 43-nt oligomer 3 degradation products are ndence should be addressed. To whom corre ndicated ex.ac.uk E-mail: AJD2 683 wwwscience mag-org SCIENCE VOL 306 22 OCTOBER 2004Explanation / Answer
1. Mt-ligase is a multidomain enzyme with functional polymerase and nuclease domains in addition to ATP dependent DNA ligase activity. In the DNA duplex, a gap is created which is 1-nt gap. The DNA duplex is 43-nt oligomer. This gap is not ligatable. This non-ligatable gap is filled by using Mt-ligase. Mt-ligase has a ATP dependent domain, so it fills the gap by attaching a phosphate group at the gap at the 5' terminus.
2. The DNA duplexes obtained will now have 3' overhangs. This DNA duplexes is then incubated with excess of Mt-ligase. The 51-nt oligomers and 43- nt ologomer substrates are degradad using Mt-ligase. There degradation products are shown in the figure B.
3. The obtained DNA substrates were incubated with large amounts of Mt-ligase. The products obtained after degradtion are indicated in the figure.
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