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DNA ligase I null mouse cells show normal DNA repair activity but altered DNA replication and reduced genome stability

¹ Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, UK
² Sir Alastair Currie CRC Laboratories, Molecular Medicine Centre, University of Edinburgh, Edinburgh EH4 2XU, UK
³ Cell Therapy Group, Scottish National Blood Transfusion Service, Edinburgh EH4 2XU, UK
⁴ John Hughes Bennett Laboratory, Department of Oncology, University of Edinburgh, Edinburgh EH4 2XU, UK

View larger version (25K): [in a new window]   Fig. 1. Structure and expression of Lig 1 targeted alleles. The structure of the 3' end of the wild-type and Lig 1 targeted alleles is shown schematically. Lig 1 exons are depicted as numbered open boxes. In the Lig 1-(#53) allele, exons 23-27 have been replaced by the PGK-HPRT(RI) Hprt minigene. Hprt exons are shown as numbered closed boxes with the Pgk promoter cross-hatched. In the Lig 1-(#12) allele, exons 23-27 have been replaced by the DWM110 Hprt minigene, with the Hprt promoter stippled. The normal RNA splicing pattern for Lig 1 mRNA is indicated and the sequences present in the Lig 1/Hprt fusion transcripts from the Lig 1 targeted alleles are shown. Note that the fusion transcript from the Lig 1-(#53) allele is rare.

View larger version (32K): [in a new window]   Fig. 2. Phenotype of Lig 1 gene targeted mice and expression from the targeted alleles. (A) Morphology of wild-type (left) and Lig 1-(#12)/-(#12) (right) embryos at E16.5. Note that the mutant embryos are smaller, anaemic and lack the erythropoiesis clearly evident in the wild-type foetal liver (*). (B) Northern analysis of Lig 1 transcripts. RNA (30 µg) extracted from primary embryonic fibroblasts (wild-type, heterozygous and homozygous for the Lig 1-(#12) and Lig 1-(#53) targeted alleles) was probed with a 2.1 kb fragment of mouse Lig 1 cDNA (Bentley et al., 1996). The position of the 3.2 kb wild-type Lig 1 mRNA is indicated. Note the larger (4.1 kb) transcript from the Lig 1-(#12) allele, whereas a transcript is undetectable from the original Lig 1-(#53) allele by northern analysis. Equivalent RNA loadings were confirmed by ethidium bromide staining of the gel prior to transfer. (C) Western analysis of DNA ligase I in wild-type and homozygous Lig 1-(#53) mutant embryos. Protein extracted from E13.5 embryos was probed with the TL5 rabbit polyclonal antibody raised against purified bovine DNA ligase I. This antibody predominantly recognises epitopes in the N-terminus of the protein. 100 µg of protein was loaded in each lane, but in addition to pure wild-type and mutant extracts, mutant extracts were also spiked with the percentages indicated of the wild-type sample. The position of the 102 kDa wild-type DNA ligase I protein is indicated by the arrow. The mobility of Mr markers (kDa) on the same gel is shown.

View larger version (34K): [in a new window]   Fig. 3. Survival of DNA-ligase-I-deficient cells exposed to the following DNA damaging agents (A) UV; (B) EMS; (C) 3-aminobenamide (3-AB); and (D) -irradiation. Mean survival±s.e.m. is shown relative to non-treated controls. , mouse wild-type (PF20); , mouse Lig1 null (PFL13); , mouse Lig1 null (PFL10); , human wild-type (MRC5); , human LIG1 point mutant (46BR); closed hexagon, mouse Ercc1 null (PF24).

View larger version (15K): [in a new window]   Fig. 4. Accumulation of DNA replication intermediates in DNA-ligase-I-deficient cells. Cultures were subjected to a short pulse with [3H]-thymidine followed by a chase of up to 30 minutes. Cells were then harvested, lysed in agarose plugs and subjected to alkaline gel electrophoresis. Fractions from <0.2 kb to >23 kb were collected and counted. The figure shows the percentage of total radioactivity present in single-stranded DNA fragments <2.0kb for each cell line. Values shown are means±s.e.m. , mouse wild-type (PF20); , human wild-type (MRC5); , mouse Lig1 null (PFL13); [UNK], human LIG1 point mutant (46BR).

View larger version (53K): [in a new window]   Fig. 5. Levels of DNA ligases III and IV are unaffected by DNA ligase I deficiency. Western analysis of protein (100 µg) extracted from mid-gestation embryos and immortalised embryonic fibroblasts. (A) Panel probed with TL25 rabbit polyclonal antibody raised against recombinant full-length human DNA ligase III. The position of the 103 kDa DNA ligase III is indicated by the arrow. (B) Panel probed with TL18 rabbit polyclonal antibody raised against a C- terminal peptide of human DNA ligase IV. The position of the 96 kDa DNA ligase IV is indicated by the arrow. The mobility of Mr markers (kDa) on the same gels is shown.

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