Different substrate specificities of the two DNA ligases of mammalian cells

Mammalian cells contain the DNA ligases I and II. These enzymes show different molecular weights and heat labilities, and antibodies against ligase I do not inhibit ligase II. Here, the nonidentical substrate specificities of the enzymes are described. Under standard reaction conditions DNA ligase I, but not ligase II, catalyzes blunt-end joining of DNA, while ligase II is the only activity that joins oligo(dT) molecules hydrogen-bonded to poly(rA). These differences facilitate the distinction between the two enzymes and should permit further analysis of their functions.     

Two distinct DNA ligases from Drosophila melanogaster embryos

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'-phosphoryl and 3'-hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg2+ for activity. The Km for ATP is 2.7 X 10(-6) M for DNA ligase I, and 3.0 X 10(-5) M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N-ethylmaleimide. DNA ligase I is active at a low salt concentration (0-30 mM KCl), but DNA ligase II is active at high salt concentrations (50-100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase-AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637-10645] seem to be the same enzyme.     

A distinct first replication cycle of DNA introduced in mammalian cells

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells.     

Exchangeability of mammalian DNA ligases between base excision repair pathways

In mammalian cells, DNA ligase IIIalpha and DNA ligase I participate in the short- and long-patch base excision repair pathways, respectively. Using an in vitro repair assay employing DNA ligase-depleted cell extracts and DNA substrates containing a single lesion repaired either through short-patch (regular abasic site) or long-patch (reduced abasic site) base excision repair pathways, we addressed the question whether DNA ligases are specific to each pathway or if they are exchangeable. We find that immunodepletion of DNA ligase I did not affect the short-patch repair pathway but blocked long-patch repair, suggesting that DNA ligase IIIalpha is not able to substitute DNA ligase I during long-patch repair. In contrast, immunodepletion of DNA ligase IIIalpha did not significantly affect either pathway. Moreover, repair of normal abasic sites in wild-type and X-ray cross-complementing gene 1 (XRCC1)-DNA ligase IIIalpha-immunodepleted cell extracts involved similar proportions of short- and long-patch repair events. This suggests that DNA ligase I was able to efficiently substitute the XRCC1-DNA ligase IIIalpha complex during short-patch repair.     

Two distinct translesion synthesis pathways across a lipid peroxidation-derived DNA adduct in mammalian cells

Translesion DNA synthesis (TLS) of damaged DNA templates is catalyzed by specialized DNA polymerases. To probe the cellular TLS mechanism, a host-vector system consisting of mouse fibroblasts and a replicating plasmid bearing a single DNA adduct was developed. This system was used to explore the TLS mechanism of a heptanone-etheno-dC (H-epsilondC) adduct, an endogenous lesion produced by lipid peroxidation. In wild-type cells, H-epsilondC almost exclusively directed incorporation of dT and dA. Whereas knockout of the Y family TLS polymerase genes, Polh, Polk, or Poli, did not qualitatively affect these TLS events, inactivation of the Rev3 gene coding for a subunit of polymerase zeta or of the Rev1 gene abolished TLS associated with dA, but not dT, insertion. The analysis of results of the cellular studies and in vitro TLS studies using purified polymerases has revealed that the insertion of dA and dT was catalyzed by different polymerases in cells. While insertion of dT can be catalyzed by polymerase eta, kappa, and iota, insertion of dA is catalyzed by an unidentified polymerase that cannot catalyze extension from the resulting dA terminus. Therefore, the extension from this terminus requires the activity of polymerase zeta-REV1. These results provide new insight into how cells use different TLS pathways to overcome a synthesis block.     

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.     

Eukaryotic DNA ligases

Recent studies on eukaryotic DNA ligases are briefly reviewed. The two distinguishable enzymes from mammalian cells, DNA ligase I and DNA ligase II, have been purified to homogeneity and characterized biochemically. Two distinct DNA ligases have also been identified in Drosophila melanogaster embryos. The genes encoding DNA ligases from Schizosaccharomyces pombe, Saccharomyces cerevisiae and vaccinia virus have been cloned and sequenced. These 3 proteins exhibit about 30% amino acid sequence identity; the 2 yeast enzymes share 53% amino acid sequence identity or conserved changes. Altered DNA ligase I activity has been found in cell lines from patients with Bloom's syndrome, although a causal link between the enzyme deficiency and the disease has not yet been proven.     

Bacterial DNA ligases

DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell. All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor. All Bacteria (eubacteria) contain NAD+-dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics. In addition to their NAD+-dependent enzymes, some Bacteria contain genes for putative ATP-dependent DNA ligases. The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism. The putative ATP-dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses. Phylogenetic analysis suggests that all NAD+-dependent DNA ligases are closely related, but the ATP-dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.     

ATP-dependent DNA ligases

SUMMARY: By catalyzing the joining of breaks in the phosphodiester backbone of duplex DNA, DNA ligases play a vital role in the diverse processes of DNA replication, recombination and repair. Three related classes of ATP-dependent DNA ligase are readily apparent in eukaryotic cells. Enzymes of each class comprise catalytic and non-catalytic domains together with additional domains of varying function. DNA ligase I is required for the ligation of Okazaki fragments during lagging-strand DNA synthesis, as well as for several DNA-repair pathways; these functions are mediated, at least in part, by interactions between DNA ligase I and the sliding-clamp protein PCNA. DNA ligase III, which is unique to vertebrates, functions both in the nucleus and in mitochondria. Two distinct isoforms of this enzyme, differing in their carboxy-terminal sequences, are produced by alternative splicing: DNA ligase IIIalpha has a carboxy-terminal BRCT domain that interacts with the mammalian DNA-repair factor XrccI, but both alpha and beta isoforms have an amino-terminal zinc-finger motif that appears to play a role in the recognition of DNA secondary structures that resemble intermediates in DNA metabolism. DNA ligase IV is required for DNA non-homologous end joining pathways, including recombination of the V(D)J immunoglobulin gene segments in cells of the mammalian immune system. DNA ligase IV forms a tight complex with Xrcc4 through an interaction motif located between a pair of carboxy-terminal BRCT domains in the ligase. Recent structural studies have shed light on the catalytic function of DNA ligases, as well as illuminating protein-protein interactions involving DNA ligases IIIalpha and IV.     

Mammalian DNA ligases

DNA joining enzymes play an essential role in the maintenance of genomic integrity and stability. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Since DNA ligase II appears to be derived from DNA ligase III by a proteolytic mechanism, the three LIG genes can account for the four biochemically distinct DNA ligase activities, DNA ligases I, II, III and IV, that have been purified from mammalian cell extracts. It is probable that the specific cellular roles of these enzymes are determined by the proteins with which they interact. The specific involvement of DNA ligase I in DNA replication is mediated by the non-catalytic amino-terminal domain of this enzyme. Furthermore, DNA ligase I participates in DNA base excision repair as a component of a multiprotein complex. Two forms of DNA ligase III are produced by an alternative splicing mechanism. The ubiqitously expressed DNA ligase III-α forms a complex with the DNA single-strand break repair protein XRCC1. In contrast, DNA ligase III-β, which does not interact with XRCC1, is only expressed in male meiotic germ cells, suggesting a role for this isoform in meiotic recombination. At present, there is very little information about the cellular functions of DNA ligase IV.     

Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation.     

Paraquat-induced DNA damage in mammalian cells

We have examined the possibility that paraquat (PQ) may exert its toxicity by inducing DNA damage. Mouse lymphoblasts in culture exhibited inhibition of colony forming ability and DNA single strand breaks following a 2 hour exposure to PQ. These phenomenon are dose dependent and increase when a rat liver S9 fraction is included in the incubation mixture. The presence of superoxide dismutase and catalase did not prevent the effects of PQ. Our data indicate that DNA should be considered as a possibile macromolecular target for the lethal effects of paraquat.     

A distinct subnuclear localization of mammalian DNA topoisomerase IIbeta in yeast

Mammalian topoisomerase II isoforms alpha and beta are diverged in their C-terminal domain (CTD), but both isoforms complement the yeast top2 mutation. In this study, mammalian topoisomerase IIalpha-CTD and IIbeta-CTD were tagged with yellow fluorescent protein (YFP), expressed in yeast cells, and their localization was examined. YFP tagged-topoisomerase IIalpha-CTD was distributed evenly throughout the nucleus, while YFP tagged-topoisomerase IIbeta-CTD was sequestered into a subnuclear compartment. Deletion analysis revealed that two regions (amino acids 1207-1234 and 1513-1573) of the topoisomerase IIbeta-CTD are essential for specific localization of the beta isoform: if either of the two regions is removed, the mutant topoisomerase IIbeta-CTD distributes evenly throughout the nucleus. The data suggest that yeast cells distinguish the nuclear and subnuclear localization signals associated with these two mammalian topoisomerase II isoforms.     

Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.