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

2003 ◽  
Vol 14 (2) ◽  
pp. 685-697 ◽  
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.

scholarly journals Human DNA Ligase IV and the Ligase IV/XRCC4 Complex:  Analysis of Nick Ligation Fidelity†

Biochemistry ◽  
2007 ◽  
Vol 46 (17) ◽  
pp. 4962-4976 ◽  
Author(s):  
Yu Wang ◽  
Brandon J. Lamarche ◽  
Ming-Daw Tsai
Keyword(s):  
Complex Analysis ◽  
Dna Ligase ◽  
Dna Ligase Iv ◽  
Human Dna ◽  
Ligase Iv

scholarly journals 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.

1995 ◽  
Vol 15 (6) ◽  
pp. 3206-3216 ◽  
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.

scholarly journals A High-Throughput Scintillation Proximity-Based Assay for Human DNA Ligase IV

2012 ◽  
Vol 10 (3) ◽  
pp. 235-249 ◽  
Author(s):  
Hui-Min Tseng ◽  
David Shum ◽  
Bhavneet Bhinder ◽  
Sindy Escobar ◽  
Nicholas J. Veomett ◽  
...  
Keyword(s):  
High Throughput ◽  
Dna Ligase ◽  
Dna Ligase Iv ◽  
Human Dna ◽  
Ligase Iv

scholarly journals Structural Insights into the Role of Domain Flexibility in Human DNA Ligase IV

Structure ◽  
2012 ◽  
Vol 20 (7) ◽  
pp. 1212-1222 ◽  
Author(s):  
Takashi Ochi ◽  
Qian Wu ◽  
Dimitri Y. Chirgadze ◽  
J. Günter Grossmann ◽  
Victor M. Bolanos-Garcia ◽  
...  
Keyword(s):  
Dna Ligase ◽  
Dna Ligase Iv ◽  
Human Dna ◽  
Ligase Iv ◽  
Structural Insights

scholarly journals Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

2015 ◽  
Vol 112 (20) ◽  
pp. E2575-E2584 ◽  
Author(s):  
Dylan A. Reid ◽  
Sarah Keegan ◽  
Alejandra Leo-Macias ◽  
Go Watanabe ◽  
Natasha T. Strande ◽  
...  
Keyword(s):  
Single Molecule ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Ligase Iv ◽  
End To End

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

scholarly journals DNA-Ligase IV and DNA-Protein Kinase Play a Critical Role in Deficient Caspases Activation in Apoptosis-resistant Cancer Cells by Using Doxorubicin

2008 ◽  
Vol 19 (8) ◽  
pp. 3283-3289 ◽  
Author(s):  
Claudia Friesen ◽  
Miriam Uhl ◽  
Ulrich Pannicke ◽  
Klaus Schwarz ◽  
Erich Miltner ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Cancer Cells ◽  
Critical Role ◽  
Dna Ligase ◽  
Apoptosis Resistance ◽  
Dsb Repair ◽  
Doxorubicin Treatment ◽  
Dna Ligase Iv ◽  
Ligase Iv

Resistance toward cytotoxic drugs is one of the primary causes for therapeutic failure in cancer therapy. DNA repair mechanisms as well as deficient caspases activation play a critical role in apoptosis resistance of tumor cells toward anticancer drug treatment. Here, we discovered that deficient caspases activation in apoptosis-resistant cancer cells depends on DNA-ligase IV and DNA-protein kinase (DNA-PK), playing crucial roles in the nonhomologous end joining (NHEJ) pathway, which is the predominant pathway for DNA double-strand break repair (DNA-DSB-repair) in mammalian cells. DNA-PK(+/+) as well as DNA-ligase IV (+/+) cancer cells were apoptosis resistant and deficient in activation of caspase-3, caspase-9, and caspase-8 and in cleavage of poly(ADP-ribose) polymerase after doxorubicin treatment. Inhibition of NHEJ by knocking out DNA-PK or DNA-ligase IV restored caspases activation and apoptosis sensitivity after doxorubicin treatment. In addition, inhibition of caspases activation prevented doxorubicin-induced apoptosis but could not prevent doxorubicin-induced DNA damage, indicating that induction of DNA damage is independent of caspases activation. However, caspases activation depends on induction of DNA damage left unrepaired by NHEJ-DNA-DSB-repair. We conclude that DNA damage left unrepaired by DNA-ligase IV or DNA-PK might be the initiator for caspases activation by doxorubicin in cancer cells. Failure in caspases activation using doxorubicin depends on loss of DNA damage and is due to higher rates of NHEJ-DNA-DBS-repair.

scholarly journals NAD+ is not utilized as a co-factor for DNA ligation by human DNA ligase IV

2020 ◽  
Vol 48 (22) ◽  
pp. 12746-12750
Author(s):  
Bailin Zhao ◽  
Tasmin Naila ◽  
Michael R Lieber ◽  
Alan E Tomkinson
Keyword(s):  
Strand Break ◽  
Dna Ligase ◽  
Unexpected Finding ◽  
Dna Ligases ◽  
Dna Ligase Iv ◽  
Human Dna ◽  
Dna Double Strand Break ◽  
Ligase Iv ◽  
Eukaryotic Dna ◽  
Catalytic Cycles

Abstract As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.

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

1995 ◽  
Vol 15 (10) ◽  
pp. 5412-5422 ◽  
Author(s):  
J Chen ◽  
A E Tomkinson ◽  
W Ramos ◽  
Z B Mackey ◽  
S Danehower ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Chromosome 17 ◽  
Dna Ligase ◽  
Cdna Clones ◽  
Dna Ligases ◽  
Cell Extracts ◽  
Human Dna ◽  
Dna Ligase I ◽  
Dna Ligase Iii

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.

scholarly journals SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV

DNA Repair ◽  
2016 ◽  
Vol 43 ◽  
pp. 18-23 ◽  
Author(s):  
George E. Greco ◽  
Yoshihiro Matsumoto ◽  
Rhys C. Brooks ◽  
Zhengfei Lu ◽  
Michael R. Lieber ◽  
...  
Keyword(s):  
Potent Inhibitor ◽  
Dna Ligase ◽  
Dna Ligase Iv ◽  
Human Dna ◽  
Ligase Iv

scholarly journals Human DNA ligase IV is able to use NAD+ as an alternative adenylation donor for DNA ends ligation

2018 ◽  
Vol 47 (3) ◽  
pp. 1321-1334 ◽  
Author(s):  
Shih-Hsun Chen ◽  
Xiaochun Yu
Keyword(s):  
Dna Ligase ◽  
Dna Ligase Iv ◽  
Human Dna ◽  
Ligase Iv ◽  
Dna Ends
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