DNA ligase-AMP adducts: Identification of yeast DNA ligase polypeptides

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [α-³²P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.     

Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.     

Vaccinia virus encodes a polypeptide with DNA ligase activity.

Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S. pombe and S. cerevisiae DNA ligases. DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction. A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP. This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate. DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R.     

DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product.

Genetic studies have previously demonstrated that the Saccharomyces cerevisiae CDC9 gene product, which is functionally homologous to mammalian DNA ligase I, is required for DNA replication and is also involved in DNA repair and genetic recombination. In the present study we have purified the yeast enzyme. When measured under denaturing conditions, Cdc9 protein has a polypeptide molecular mass of 87 kDa. The native form of the enzyme is an 80-kDa asymmetric monomer. Both estimates are in good agreement with the M(r) = 84,406 predicted from the translated sequence of the CDC9 gene. Cdc9 DNA ligase acts via the same basic reaction mechanism employed by all known ATP-dependent DNA ligases. The catalytic functions reside in a 70-kDa C-terminal domain that is conserved in mammalian DNA ligase I and in Cdc17 DNA ligase from Schizosaccharomyces pombe. The ATP analog ATP alpha S inhibits the ligation reaction, although Cdc9 protein does form an enzyme-thioadenylate intermediate. Since Cdc9 DNA ligase exhibited the same substrate specificity as mammalian DNA ligase I, this enzyme can be considered to be the DNA ligase I of S. cerevisiae. There is genetic evidence suggesting that DNA ligase may be directly involved in error-prone DNA repair. We examined the ability of Cdc9 DNA ligase to join nicks with mismatches at the termini. Mismatches at the 5' termini of nicks had very little effect on ligation, whereas mismatches opposite a purine at 3' termini inhibited DNA ligation. The joining of DNA molecules with mismatched termini by DNA ligase may be responsible for the generation of mutations.     

Mammalian DNA ligases. Biosynthesis and intracellular localization of DNA ligase I

Mammalian DNA ligase I is presumed to act in DNA replication. Rabbit antibodies against the homogeneous enzyme from calf thymus inhibited DNA ligase I activity and consistently recognized a single polypeptide of 125 kDa when cells from an established bovine kidney cell line (MDBK) were lysed rapidly by a variety of procedures and subjected to immunoblotting analysis. After biosynthetic labeling of MDBK cells with [35S]methionine, immunoprecipitation experiments revealed a polypeptide of 125 kDa that did not appear when purified calf thymus DNA ligase I was used in competition. A 125-kDa polypeptide was adenylated when immunoprecipitated protein from MDBK cells was incubated with [alpha-32P]ATP. Thus, the apparent molecular mass of the initial translation product is identical or nearly so to that of the purified enzyme. The half-life of the protein is 7 h as determined by pulse-chase experiments in asynchronous MDBK cells. Immunocytochemistry and indirect immunofluorescence experiments showed that DNA ligase I is localized to cell nuclei.     

Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.     

Vaccinia DNA ligase complements Saccharomyces cerevisiae cdc9, localizes in cytoplasmic factories and affects virulence and virus sensitivity to DNA damaging agents.

The functional compatibility of vaccinia virus DNA ligase with eukaryotic counterparts was demonstrated by its ability to complement Saccharomyces cerevisiae cdc9. The vaccinia DNA ligase is a 63 kDa protein expressed early during infection that is non-essential for virus DNA replication and recombination in cultured cells. This implies complementation by a mammalian DNA ligase, yet no obvious recruitment of host DNA ligase I from the nucleus to the cytoplasm was observed during infection. An antiserum raised against a peptide conserved in eukaryotic DNA ligases identified the virus enzyme in discrete cytoplasmic 'factories', the sites of virus DNA synthesis, demonstrating immunological cross-reactivity between host DNA ligase I and the vaccinia enzyme. DNA ligase was not detected in the factories of a mutant virus lacking the ligase gene. Despite this, no difference in growth between wild-type (WT) and mutant virus was detectable even in Bloom's syndrome cells which have reduced DNA ligase I activity. However, DNA ligase negative virus showed an increased sensitivity to UV or bleomycin in cultured cells, and the importance of DNA ligase for virus virulence in vivo was demonstrated by the attenuated phenotype of the deletion mutant in intranasally infected mice.     

An African swine fever virus gene with homology to DNA ligases.

Sequence analysis of the SalI g region of the genome of a virulent isolate of ASFV (Malawi Lil 20/1) has revealed an open reading frame with the potential to encode a 48 kilodalton (kD) polypeptide which has significant homology with eukaryotic and prokaryotic DNA ligases. This ASFV encoded gene also contains the putative active site region of DNA ligases including the lysine residue which is necessary for enzyme-adenylate adduct formation, but lacks the C-terminal basic region conserved in other eukaryotic DNA ligases. A novel [32P]-labelled potential DNA ligase-adenylate adduct of M(r) 45 kD was observed upon incubation of ASFV infected cell cytoplasmic extracts with alpha-[32P]-ATP and subsequent analysis of products by SDS/PAGE. These data together suggest that ASFV encodes its own DNA ligase.     

Reinvestigation of DNA ligase I in axolotl and Pleurodeles development.

We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules.     

Defective DNA ligase I in Bloom's syndrome cells. Simultaneous analysis using immunoblotting and the ligase-[32P]AMP adduct assay

Cells from patients with Bloom's syndrome (BS), an autosomal recessive disorder associated with an increased risk of cancer, exhibit genomic instability. Increased numbers of sister-chromatid exchanges (SCE) and delayed DNA chain maturation are typically observed in BS cells. To elucidate the basis for the previously reported decreased DNA ligase I activity in BS cells, simultaneous immunoblot and activity assays for ligase-[32P]AMP adduct formation were performed on extracts from BS and normal lymphoblastoid cell lines. Immunoblot analysis using antibody to DNA ligase I indicate that the amount of the major reactive protein (98 kDa) in normal and BS cells is similar. However, a 50-90% decrease was observed in the ligase activity of the 98-kDa polypeptide in high-SCE BS cells (HG1514 and GM3403c). In contrast, the activity in low-SCE BS cells (HG1554) did not differ significantly from that in normal cells. The data, together with mixing experiments, indicate that the defect in BS ligase I is due at least in part to the loss of ATP binding and/or hydrolytic activity and not to differences in numbers of protein molecules or inhibitory substances. These results suggest that mutation of the DNA ligase I gene may account for the primary metabolic defect in BS.     

Immunochemical analysis of molecular forms of mammalian DNA ligases I and II

Using specific antibodies against calf thymus DNA ligases I and II (EC 6.5.1.1), we have investigated the polypeptide structures of DNA ligases I and II present in the impure enzyme preparations, and estimated the polypeptides of DNA ligases I and II present in vivo. Immunoblot analysis of DNA ligase I after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 130-kDa polypeptide as a major one in the enzyme preparations from calf thymus throughout the purification. In addition to the 130-kDa polypeptide, a 200-kDa polypeptide was detected in the enzyme preparations at the earlier steps of the purification, and a 90-kDa polypeptide was observed as a minor one in the enzyme preparations at the later steps of the purification. The polypeptides with molecular weight of 130 000 and 90 000 were detected by SDS-polyacrylamide gel electrophoresis of DNA ligase I-[3H]AMP complex. These results suggest that a 200-kDa polypeptide of DNA ligase I present in vivo is degraded to a 130-kDa polypeptide and then to a 90-kDa polypeptide during the isolation and purification procedures. On the other hand, the monospecific antibody against calf thymus DNA ligase II cross-reacted with only a 68 kDa polypeptide in the enzyme preparations throughout the purification, suggesting that the 68-kDa polypeptide is a single form of calf thymus DNA ligase II present in vivo as well as in vitro.     

The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae.

DNA polymerase III (delta) of Saccharomyces cerevisiae is purified as a complex of at least two polypeptides with molecular masses of 125 and 55 kDa as judged by SDS-PAGE. In this paper we determine partial amino acid sequences of the 125 and 55 kDa polypeptides and find that they match parts of the amino acid sequences predicted from the nucleotide sequence of the CDC2 and HYS2 genes respectively. We also show by Western blotting that Hys2 protein co-purifies with DNA polymerase III activity as well as Cdc2 polypeptide. The complex form of DNA polymerase III activity could not be detected in thermosensitive hys2 mutant cell extracts, although another form of DNA polymerase III was found. This form of DNA polymerase III, which could also be detected in wild-type extracts, was not associated with Hys2 protein and was not stimulated by addition of proliferating cell nuclear antigen (PCNA), replication factor A (RF-A) or replication factor C (RF-C). The temperature-sensitive growth phenotype of hys2-1 and hys2-2 mutations could be suppressed by the CDC2 gene on a multicopy plasmid. These data suggest that the 55 kDa polypeptide encoded by the HYS2 gene is one of the subunits of DNA polymerase III complex in S.cerevisiae and is required for highly processive DNA synthesis catalyzed by DNA polymerase III in the presence of PCNA, RF-A and RF-C.     

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.     

Arabidopsis DNA ligase IV is induced by gamma-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4

Rejoining of single- and double-strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double-strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by gamma-irradiation, represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP-dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino-acid sequence to the human DNA ligase IV. Yeast two-hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV-binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C-terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by gamma-irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.     

Mitochondrial DNA ligase function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4). The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.     

The gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae

Abstract We report the isolation, genomic mapping, and DNA sequence of the BPL1 gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae . The gene was isolated by complementation of an Escherichia coli birA (biotin-apoprotein ligase) mutant indicating that the expressed yeast protein modified the essential biotinated protein of the bacterial host. The BPL1 gene encodes a protein of 690 residues ( M _r 76.4 kDa) with strong sequence similarites to the E. coli and human biotin-apoprotein ligases. BPL1 was mapped to chromosome IV, is allelic to the previously described ACC2 gene, and encodes the major (if not the only) biotin-apoprotein ligase activity of S. cerevisiae .     

The small subunits of human and mouse DNA polymerase epsilon are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase epsilon.

Human DNA polymerase epsilon is composed of a 261 kDa catalytic polypeptide and a 55 kDa small subunit of unknown function. cDNAs encoding the small subunit of human and mouse DNA polymerase epsilon were cloned. The predicted polypeptides have molecular masses of 59.469 and 59.319 kDa respectively and they are 90% identical. The human and mouse polypeptides show 22% identity with the 80 kDa subunit of the five subunit DNA polymerase epsilon from the yeast Saccharomyces cerevisiae. The high degree of conservation suggests that the 55 kDa subunit shares an essential function with the yeast 80 kDa subunit, which was earlier suggested to be involved in S phase cell cycle control in a pathway that is able to sense and signal incomplete replication. The small subunits of human and mouse DNA polymerase epsilon also show homology to the C-terminal domain of the second largest subunit of DNA polymerase alpha. The gene for the small subunit of human DNA polymerase epsilon (POLE2) was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.     

Biosynthesis of mammalian DNA ligase

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.