Relationship between the S. cerevisiae LIG4 gene product and those of other eukaryotic ATP-dependent DNA ligases. Sources of proteins compared are caLIG (C. albicans CDC9 EMBL accession no. X95001); scLIG4 (S. cerevisiae LIG4, SGD, YOR005C); hsLIG4 (Homo sapiens DNA ligase IV, EMBL accession no. X83441); hsLIG1 (Homo sapiens DNA ligase I, GenBank accession no. M36067); scCDC9 (S. cerevisiae CDC9, EMBL accession no. X03246); hsLIG3 (Homo sapiens DNA ligase III, EMBL accession no. X84740). The amino acid sequences were aligned and the phylogenetic relationship calculated with the PILEUP and BESTFIT programs of the Genetic Computer Group (Devereux et al. 1984); gap weight, 5; gap length weight, 0.1. (A) Phylogenetic relationship of the scLIG4 protein to eukaryotic DNA ligase I, III, and IV type enzymes. (B) Amino acid sequence alignment of the yeast and human DNA ligase I and IV type enzymes. Identical residues are boxed. Shaded boxes denote positions of sequence motifs that are highly conserved between all ATP-dependent DNA ligases. The position of the adenylated lysine residue is residue 282 in scLIG4. Underscored are the carboxy-terminal regions that define the BRCT domains as identified by hydrophobic cluster analysis.

Recombinant scLIG4 protein shows the catalytic properties of an ATP-dependent DNA ligase. (A) Co-purification of histidine-tagged scLIG4 protein with enzyme-adenylate forming activity. The left panel (lanes 1–6) shows the protein content in relevant nickel column fractions (17 μl aliquots), by silver-staining after SDS–polyacrylamide gel electrophoresis (7.5%). The right panel (lanes 7–12) shows the same fractions (4 μl aliquots) assayed for enzyme–AMP complex formation as revealed after autoradiography. (Lanes 1,7) First 80 mm imidazole wash; (lanes 2,8) second 80 mm imidazole wash; (lanes 3,9) first 500 mm imidazole elution; (lanes 4,10) second 500 mm imidazole elution; (lanes 5,11) third 500 mm imidazole elution; (lanes 6,12) second 500 mm imidazole elution (corresponding to lanes 4 and 10) of a parallel vector control purification from E. coli BL21(DE3) cells with the pET16b vector alone. (B) Formation of DNA–adenylate intermediate. Reactions were carried out as described in Materials and Methods, and products were analyzed by denaturing polyacrylamide gel electrophoresis. (Lane 1) ³²P-labeled molecular mass standard (dT)₁₆; (lane 2), 1 mU of T4 DNA ligase incubated with oligo (dT)₁₆ ⋅ poly(rA); (lanes 3–7) 6 μl of scLIG4 incubated with oligo (dT)₁₆ ⋅ poly(rA); [5′-OH] oligo (dT)₁₆ ⋅ poly(rA); oligo (dT)₁₆ ⋅ poly(dA); [5′-OH] oligo (dT)₁₆ ⋅ poly(dA); and oligo (dT)₁₆, respectively. (C) Ligation of polynucleotide substrates. Reactions were carried out as described in Materials and Methods. Lanes 1–4 show ligation assayed with [5-³²P] oligo (dT)₁₆ ⋅ poly(rA), lanes 5–8 with [5-³²P] oligo (dT)₁₆ ⋅ poly(dA). Enzymes were added as follows; (lanes 1,5) T4 DNA ligase controls (1 mU and 0.1 mU, respectively); (lanes 2,6) no enzyme; (lanes 3,7) 6 μl scLIG4; (lanes 4,8) 6 μl of corresponding vector control fractions.

S. cerevisiae lig4 mutant cells are deficient in recircularizing linearized plasmid DNA. Plasmid transformation assays were carried out as detailed in Materials and Methods using either supercoiled, EcoRI-digested, or SmaI-digested pBTM116 plasmid DNA for transformation. (A) Schematic map of the yeast replicative plasmid pBTM116, carrying Amp for selection in E. coli and the yeast TRP1 gene as a selectable yeast marker. A multiple cloning sequence including the relevant EcoRI and SmaI sites is located within a stretch of DNA sequence which shares no homology with S. cerevisiae genomic sequences. This DNA is flanked by transcription control elements of yeast ADH1 (pADH1, tADH1). Numbers below the map indicate the distance in base pairs of relevant plasmid elements from the EcoRI cut site, and the arrows mark the annealing sites of the PCR primers used for analysis of plasmid repair events. (B) Graphic illustration of relative transformation efficiencies (ratios cut/uncut plasmids) obtained with EcoRI- (left) and SmaI-cut plasmid. Data are averages from at least three independent transformation experiments in which matching test and control strains were treated in parallel. Strains used LIG4, FF18734; lig4, PRSY003,1; rad52, FF18743; lig4 rad52; PRSY005; lig4/pLIG4, PRSY003,1 carrying a LIG4-expressing plasmid (pPRS156); lig4/pYES2, PRSY003,1 carrying the expression vector only.

Sensitivity of lig4 mutant cells to DNA damaging agents. Cells were exposed to UV, MMS, or ionizing radiation as indicated. Survival data are shown from at least three independent experiments. Standard deviations are shown where relevant for judgment of differences between strains. Matching test and control strains were always assayed in parallel as detailed in Materials and Methods. The strains used are listed with genotypes in Table 2 and are referred to in the figure as (wt) FF18734 and FF18984 (▪); (lig4) PRSY003,1 and PRSY004,2 (□); (rad52) FF18743 (♦); (lig4 rad52) PRSY005, PRSY006 (⋄); (cdc9) PRSY009 (•); and (cdc9 lig4) PRSY011 (○).

LIG4-deficient diploids are delayed in meiosis I. Fractions of cells in sporulating populations that have passed meiosis I, as evidenced by DAPI staining, are shown as a function of time postinduction. Analyses were performed as described in Materials and Methods and averages from two independent experiments are given. The values represent accumulated fractions of cells with two or more DAPI staining bodies in the sporulating populations. Strains are referred to as follows: (LIG4/LIG4) diploid FF18734/FF18984 (▪); (LIG4/lig4) diploids FF18734/PRSY004,2 and PRSY003,1/FF18984 (□); (lig4/lig4) diploid PRSY003,1/PRSY004,2 (♦).