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.

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Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3206 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3206-3216 ◽

Cited By ~ 164

Author(s):

Y F Wei ◽

P Robins ◽

K Carter ◽

K Caldecott ◽

D J Pappin ◽

...

Keyword(s):

Dna Repair ◽

Cdna Clone ◽

Dna Ligase ◽

Dna Ligases ◽

Dna Ligase Iv ◽

Ligase Iv ◽

Dna Ligase I ◽

Cloned Cdna ◽

Dna Ligase Iii

Three distinct DNA ligases, I to III, have been found previously in mammalian cells, but a cloned cDNA has been identified only for DNA ligase I, an essential enzyme active in DNA replication. A short peptide sequence conserved close to the C terminus of all known eukaryotic DNA ligases was used to search for additional homologous sequences in human cDNA libraries. Two different incomplete cDNA clones that showed partial homology to the conserved peptide were identified. Full-length cDNAs were obtained and expressed by in vitro transcription and translation. The 103-kDa product of one cDNA clone formed a characteristic complex with the XRCC1 DNA repair protein and was identical with the previously described DNA ligase III. DNA ligase III appears closely related to the smaller DNA ligase II. The 96-kDa in vitro translation product of the second cDNA clone was also shown to be an ATP-dependent DNA ligase. A fourth DNA ligase (DNA ligase IV) has been purified from human cells and shown to be identical to the 96-kDa DNA ligase by unique agreement between mass spectrometry data on tryptic peptides from the purified enzyme and the predicted open reading frame of the cloned cDNA. The amino acid sequences of DNA ligases III and IV share a related active-site motif and several short regions of homology with DNA ligase I, other DNA ligases, and RNA capping enzymes. DNA ligases III and IV are encoded by distinct genes located on human chromosomes 17q11.2-12 and 13q33-34, respectively.

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Identification of a novel human DNA ligase I inhibitor that promotes cellular apoptosis in DLD-1 cells: an in silico and in vitro mechanistic study

RSC Advances ◽

10.1039/c6ra22364h ◽

2016 ◽

Vol 6 (97) ◽

pp. 94574-94587 ◽

Cited By ~ 3

Author(s):

Deependra Kumar Singh ◽

Mohd. Kamil Hussain ◽

Shagun Krishna ◽

Amit Laxmikant Deshmukh ◽

Mohammad Shameem ◽

...

Keyword(s):

In Silico ◽

Specific Inhibitor ◽

Mechanistic Study ◽

Dna Ligase ◽

Dna Ligases ◽

Human Dna ◽

Dna Ligase I ◽

Cellular Apoptosis

The compound S-097/98 is a specific inhibitor of hLig1. As shown in the figure, the compound inhibits only hLig1 while other human and non-human DNA ligases are not inhibited.

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The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates

Nucleic Acids Research ◽

10.1093/nar/gkaa151 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3708-3721 ◽

Cited By ~ 4

Author(s):

Melike Çağlayan

Keyword(s):

Excision Repair ◽

Dna Ligase ◽

Active Site Residues ◽

Dna Ligases ◽

Terminal Domain ◽

Dna Ligase I ◽

Nucleotide Insertion ◽

Base Excision ◽

Ligation Step

Abstract DNA ligase I and DNA ligase III/XRCC1 complex catalyze the ultimate ligation step following DNA polymerase (pol) β nucleotide insertion during base excision repair (BER). Pol β Asn279 and Arg283 are the critical active site residues for the differentiation of an incoming nucleotide and a template base and the N-terminal domain of DNA ligase I mediates its interaction with pol β. Here, we show inefficient ligation of pol β insertion products with mismatched or damaged nucleotides, with the exception of a Watson–Crick-like dGTP insertion opposite T, using BER DNA ligases in vitro. Moreover, pol β N279A and R283A mutants deter the ligation of the promutagenic repair intermediates and the presence of N-terminal domain of DNA ligase I in a coupled reaction governs the channeling of the pol β insertion products. Our results demonstrate that the BER DNA ligases are compromised by subtle changes in all 12 possible noncanonical base pairs at the 3′-end of the nicked repair intermediate. These findings contribute to understanding of how the identity of the mismatch affects the substrate channeling of the repair pathway and the mechanism underlying the coordination between pol β and DNA ligase at the final ligation step to maintain the BER efficiency.

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In Vitro and In Vivo Interactions of DNA Ligase IV with a Subunit of the Condensin Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e01-11-0117 ◽

2003 ◽

Vol 14 (2) ◽

pp. 685-697 ◽

Cited By ~ 18

Author(s):

Marcin R. Przewloka ◽

Paige E. Pardington ◽

Steven M. Yannone ◽

David J. Chen ◽

Robert B. Cary

Keyword(s):

Molecular Mechanisms ◽

Critical Role ◽

Dna Ligase ◽

Mitotic Chromosomes ◽

Dna Ligase Iv ◽

Cell Extracts ◽

Condensin Complex ◽

Human Dna ◽

Ligase Iv

Several findings have revealed a likely role for DNA ligase IV, and interacting protein XRCC4, in the final steps of mammalian DNA double-strand break repair. Recent evidence suggests that the human DNA ligase IV protein plays a critical role in the maintenance of genomic stability. To identify protein–protein interactions that may shed further light on the molecular mechanisms of DSB repair and the biological roles of human DNA ligase IV, we have used the yeast two-hybrid system in conjunction with traditional biochemical methods. These efforts have resulted in the identification of a physical association between the DNA ligase IV polypeptide and the human condensin subunit known as hCAP-E. The hCAP-E polypeptide, a member of the Structural Maintenance of Chromosomes (SMC) super-family of proteins, coimmunoprecipitates from cell extracts with DNA ligase IV. Immunofluorescence studies reveal colocalization of DNA ligase IV and hCAP-E in the interphase nucleus, whereas mitotic cells display colocalization of both polypeptides on mitotic chromosomes. Strikingly, the XRCC4 protein is excluded from the area of mitotic chromosomes, suggesting the formation of specialized DNA ligase IV complexes subject to cell cycle regulation. We discuss our findings in light of known and hypothesized roles for ligase IV and the condensin complex.

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Kinetic analyses of single-stranded break repair by human DNA ligase III isoforms reveal biochemical differences from DNA ligase I

Journal of Biological Chemistry ◽

10.1074/jbc.m117.804625 ◽

2017 ◽

Vol 292 (38) ◽

pp. 15870-15879 ◽

Cited By ~ 4

Author(s):

Justin R. McNally ◽

Patrick J. O'Brien

Keyword(s):

Dna Ligase ◽

Human Dna ◽

Dna Ligase I ◽

Break Repair ◽

Dna Ligase Iii

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Tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can specifically target bacterial DNA ligases and can distinguish them from human DNA ligase I

Organic & Biomolecular Chemistry ◽

10.1039/c5ob00439j ◽

2015 ◽

Vol 13 (19) ◽

pp. 5475-5487 ◽

Cited By ~ 6

Author(s):

Nisha Yadav ◽

Taran Khanam ◽

Ankita Shukla ◽

Niyati Rai ◽

Kanchan Hajela ◽

...

Keyword(s):

Dna Ligase ◽

Indole Derivatives ◽

Dna Metabolism ◽

Bacterial Dna ◽

Dna Ligases ◽

Human Dna ◽

Dna Ligase I ◽

Critical Components

DNA ligases are critical components for DNA metabolism in all organisms.

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Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.310-317.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 310-317

Author(s):

C Prigent ◽

M S Satoh ◽

G Daly ◽

D E Barnes ◽

T Lindahl

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Excision Repair ◽

Residual Activity ◽

Dna Ligase ◽

Missense Mutations ◽

Cell Extracts ◽

Dna Ligase I ◽

Enzymatic Defect

Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A subline of identical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially active enzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% of normal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nicked DNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion of nicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNA replication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation of Okazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragments remain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extracts shows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purified normal DNA ligase I.

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