Completion of base excision repair by mammalian DNA ligases.

Three mammalian genes encoding DNA ligases--LIG1, LIG3, and LIG4--have been identified. Genetic, biochemical, and cell biology studies indicate that the products of each of these genes play a unique role in mammalian DNA metabolism. Interestingly, cell lines deficient in either DNA ligase I (46BR.1G1) or DNA ligase III (EM9) are sensitive to simple alkylating agents. One interpretation of these observations is that DNA ligases I and III participate in functionally distinct base excision repair (BER) subpathways. In support of this idea, extracts from both DNA ligase-deficient cell lines are defective in catalyzing BER in vitro and both DNA ligases interact with other BER proteins. DNA ligase I interacts directly with proliferating cell nuclear antigen (PCNA) and DNA polymerase beta (Pol beta), linking this enzyme with both short-patch and long-patch BER. In somatic cells, DNA ligase III alpha forms a stable complex with the DNA repair protein Xrcc1. Although Xrcc1 has no catalytic activity, it also interacts with Pol beta and poly(ADP-ribose) polymerase (PARP), linking DNA ligase III alpha with BER and single-strand break repair, respectively. Biochemical studies suggest that the majority of short-patch base excision repair events are completed by the DNA ligase III alpha/Xrcc1 complex. Although there is compelling evidence for the participation of PARP in the repair of DNA single-strand breaks, the role of PARP in BER has not been established.

DNA ligase III is recruited to DNA strand breaks by a zinc finger motif homologous to that of poly(ADP-ribose) polymerase. Identification of two functionally distinct DNA binding regions within DNA ligase III.

Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activity in vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivo the DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.

Structure and function of mammalian DNA ligases.

DNA joining events are required for the completion of DNA replication, DNA excision repair and genetic recombination. Five DNA ligase activities, I-V, have been purified from mammalian cell extracts and three mammalian LIG genes, LIG1 LIG3 and LIG4, have been cloned. During DNA replication, the joining of Okazaki fragments by the LIG1 gene product appears to be mediated by an interaction with proliferating cell nuclear antigen (PCNA). This interaction may also occur during the completion of mismatch, nucleotide excision and base excision repair (BER). In addition, DNA ligase I participates in a second BER pathway that is carried out by a multiprotein complex in which DNA ligase I interacts directly with DNA polymerase beta. DNA ligase III alpha and DNA ligase III beta, which are generated by alternative splicing of the LIG3 gene, can be distinguished by their ability to bind to the DNA repair protein, XRCC1. The interaction between DNA ligase III alpha and XRCC1, which occurs through BRCT motifs in the C-termini of these polypeptides, implicates this isoform of DNA ligase III in the repair of DNA single-strand breaks and BER. DNA ligase II appears to be a proteolytic fragment of DNA ligase III alpha. The restricted expression of DNA ligase III beta suggests that this enzyme may function in the completion of meiotic recombination or in a postmeiosis DNA repair pathway. Complex formation between DNA ligase IV and the DNA repair protein XRCC4 involves the C-terminal region of DNA ligase IV, which contains two BRCT motifs. This interaction, which stimulates DNA joining activity, implies that DNA ligase IV functions in V(D)J recombination and non-homologous end-joining of DNA double-strand breaks. At the present time, it is not known whether DNA ligase V is derived from one of the known mammalian LIG genes or is the product of a novel gene.

An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.

Role of deoxyribonucleic acid polymerase epsilon in spermatogenesis in mice.

Previous studies on DNA polymerase epsilon indicate that this enzyme is involved in replication of chromosomal DNA. In this study, we examined the expression of DNA polymerases alpha, delta, and epsilon during mouse testis development and germ cell differentiation. The steady-state levels of mRNAs encoding DNA polymerase epsilon and the recombination enzyme Rad51 remained constant during testis development, whereas the mRNA levels of DNA polymerases alpha and delta declined from birth until sexual maturity. Immunohistochemical staining methods, using a stage-specific model of the seminiferous epithelium, revealed dramatic differences between DNA polymerase alpha and epsilon distribution. As expected, DNA polymerase alpha and proliferating cell nuclear antigen showed relatively strong immunostaining in mitotically proliferating spermatogonia and even stronger staining in preleptotene cells undergoing meiotic DNA replication. The distribution of Rad51 was similar, but there was a dramatic peak in late pachytene cells. In contrast, DNA polymerase epsilon was detectable in mitotically proliferating spermatogonia but not in the early stages of meiotic prophase. However, DNA polymerase epsilon reappeared in late pachytene cells and remained through the two meiotic divisions, and was present in haploid spermatids up to the stage at which the flagellum starts developing. Overall, the results suggest that DNA polymerase epsilon functions in mitotic replication, in the completion of recombination in late pachytene cells, and in repair of DNA damage in round spermatids. In contrast, DNA polymerases alpha and delta appear to be involved in meiotic DNA synthesis, which occurs early in meiotic prophase, in addition to functioning in DNA replication in proliferating spermatogonia.

Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.

Purification and characterization of DNA ligase III from bovine testes. Homology with DNA ligase II and vaccinia DNA ligase.

Mammalian cell nuclei contain three biochemically distinct DNA ligases. In the present study we have found high levels of DNA ligase I and DNA ligase III activity in bovine testes and have purified DNA ligase III to near homogeneity. The high level of DNA ligase III suggests a role for this enzyme in meiotic recombination. In assays measuring the fidelity of DNA joining, we detected no significant differences between DNA ligases II and III, whereas DNA ligase I was clearly a more faithful enzyme and was particularly sensitive to 3' mismatches. Amino acid sequences of peptides derived from DNA ligase III demonstrated that this enzyme, like DNA ligase II, is highly homologous with vaccinia DNA ligase. The absence of unambiguous differences between homologous peptides from DNA ligases II and III (10 pairs of peptides, 136 identical amino acids) indicates that these enzymes are either derived from a common precursor polypeptide or are encoded from the same gene by alternative splicing. Based on similarities in amino acid sequence and biochemical properties, we suggest that DNA ligases II and III, Drosophila DNA ligase II, and the DNA ligases encoded by the pox viruses constitute a distinct family of DNA ligases that perform specific roles in DNA repair and genetic recombination.

Mammalian DNA ligase II is highly homologous with vaccinia DNA ligase. Identification of the DNA ligase II active site for enzyme-adenylate formation.

Mammalian cells contain three biochemically distinct DNA ligases. In this report we describe the purification of DNA ligase II to homogeneity from bovine liver nuclei. This enzyme interacts with ATP to form an enzyme-AMP complex, in which the AMP moiety is covalently linked to a lysine residue. An adenylylated peptide from DNA ligase II contains the sequence, Lys-Tyr-Asp-Gly-Glu-Arg, which is homologous to the active site motif conserved in ATP-dependent DNA ligases. The sequences adjacent to this motif in DNA ligase II are different from the comparable sequences in DNA ligase I, demonstrating that these enzymes are encoded by separate genes. The amino acid sequences of 15 DNA ligase II peptides exhibit striking homology (65% overall identity) with vaccinia DNA ligase. These peptides are also homologous (31% overall identity) with the catalytic domain of mammalian DNA ligase I, indicating that the genes encoding DNA ligases I and II probably evolved from a common ancestral gene. Since vaccinia DNA ligase is not required for DNA replication but influences the ability of the virus to survive DNA damage, the homology between this enzyme and DNA ligase II suggests that DNA ligase II may be involved in DNA repair.