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

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.

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

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.

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

2020 ◽  
Vol 48 (7) ◽  
pp. 3708-3721 ◽  
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.

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 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.

DNA ligase I null mouse cells show normal DNA repair activity but altered DNA replication and reduced genome stability

2002 ◽  
Vol 115 (7) ◽  
pp. 1551-1561 ◽  
Author(s):  
Darren J. Bentley ◽  
Caroline Harrison ◽  
Ann-Marie Ketchen ◽  
Nicola J. Redhead ◽  
Kay Samuel ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Genome Instability ◽  
Null Alleles ◽  
Dna Ligase ◽  
Null Mouse ◽  
Dna Ligase I ◽  
Mouse Cells

DNA ligase I is the key ligase for DNA replication in mammalian cells and has also been reported to be involved in a number of recombination and repair processes. Our previous finding that Lig1 knockout mouse embryos developed normally to mid-term before succumbing to a specific haematopoietic defect was difficult to reconcile with a report that DNA ligase I is essential for the viability of cultured mammalian cells. To address this issue, we generated a second Lig1 targeted allele and found that the phenotypes of our two Lig1 mutant mouse lines are identical. Widely different levels of Lig1 fusion transcripts were detected from the two targeted alleles, but we could not detect any DNA ligase I protein, and we believe both are effective Lig1 null alleles. Using foetal liver cells to repopulate the haematopoietic system of lethally irradiated adult mice, we demonstrate that the haematopoietic defect in DNA-ligase-I-deficient embryos is a quantitative deficiency relating to reduced proliferation rather than a qualitative block in any haematopoietic lineage. DNA ligase I null fibroblasts from Lig1 mutant embryos showed an accumulation of DNA replication intermediates and increased genome instability. In the absence of a demonstrable deficiency in DNA repair we postulate that, unusually, genome instability may result directly from the DNA replication defect. Lig1null mouse cells performed better in the survival and replication assays than a human LIG1 point mutant, and we suggest that the complete absence of DNA ligase I may make it easier for another ligase to compensate for DNA ligase I deficiency.

scholarly journals DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized nucleotide insertion products and base excision repair intermediates with mismatches

2020 ◽  
Author(s):  
Pradnya Kamble ◽  
Kalen Hall ◽  
Mahesh Chandak ◽  
Qun Tang ◽  
Melike Çağlayan
Keyword(s):  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Ligase ◽  
Base Pairs ◽  
Processing Enzymes ◽  
Dna Ligase I ◽  
Nucleotide Insertion ◽  
Base Excision

ABSTRACTDNA ligase I (LIG1) completes base excision repair (BER) pathway at the last nick sealing step following DNA polymerase (pol) β gap filling DNA synthesis. We previously reported that pol β 8-oxo-2’-deoxyribonucleoside 5’-triphosphate (8-oxodGTP) insertion confounds LIG1 leading to the formation of ligation failure products with 5’-adenylate (AMP) block. Here, we report the mutagenic ligation of pol β 8-oxodGTP insertion products and an inefficient substrate-product channeling from pol β Watson-Crick like dG:T mismatch insertion to DNA ligation by LIG1 mutant with perturbed fidelity (E346A/E592A) in vitro. Moreover, our results revealed that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3’-8-oxodG or mismatches is governed by the mutations at both E346 and E592 residues. Finally, we found that Aprataxin (APTX) and Flap Endonuclease 1 (FEN1), as compensatory DNA-end processing enzymes, can remove 5’-AMP block from the abortive ligation products with 3’-8-oxodG or all possible 12 non-canonical base pairs. These findings contribute to understand the role of LIG1 as an important determinant of faithful BER, and how a multi-protein complex (LIG1, pol β, APTX and FEN1) can coordinate to hinder the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway.

scholarly journals The human checkpoint sensor and alternative DNA clamp Rad9–Rad1–Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery

2005 ◽  
Vol 389 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Ekaterina SMIRNOVA ◽  
Magali TOUEILLE ◽  
Enni MARKKANEN ◽  
Ulrich HÜBSCHER
Keyword(s):  
Quality Control ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Ligase ◽  
Dna Polymerase Β ◽  
Flap Endonuclease 1 ◽  
Important Target ◽  
Dna Ligase I ◽  
Base Excision

The human checkpoint sensor and alternative clamp Rad9–Rad1–Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9–Rad1–Hus1 with MutY DNA glycosylase, DNA polymerase β and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9–Rad1–Hus1 complex, thus enhancing the quality control of DNA.

scholarly journals UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

2001 ◽  
Vol 12 (5) ◽  
pp. 1199-1213 ◽  
Author(s):  
Gregory G. Oakley ◽  
Lisa I. Loberg ◽  
Jiaqin Yao ◽  
Mary A. Risinger ◽  
Remy L. Yunker ◽  
...  
Keyword(s):  
Dna Replication ◽  
Uv Radiation ◽  
Excision Repair ◽  
Genetic Disorder ◽  
Protein A ◽  
Dna Recombination ◽  
Delayed Response ◽  
Replication Intermediates

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

scholarly journals An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76 ◽  
Author(s):  
K W Caldecott ◽  
C K McKeown ◽  
J D Tucker ◽  
S Ljungquist ◽  
L H Thompson
Keyword(s):  
Dna Repair ◽  
Affinity Chromatography ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Affinity Purification ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Dna Ligase ◽  
Base Excision ◽  
Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro

1992 ◽  
Vol 12 (7) ◽  
pp. 3041-3049
Author(s):  
L Bardwell ◽  
A J Cooper ◽  
E C Friedberg
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Specific Interaction ◽  
Mitotic Recombination ◽  
Nucleotide Excision ◽  
Specific Association ◽  
Recombination Pathway ◽  
Cloned Gene

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are two of at least seven genes which are known to be required for damage-specific recognition and/or damage-specific incision of DNA during nucleotide excision repair. RAD1 and RAD10 are also involved in a specialized mitotic recombination pathway. We have previously reported the purification of the RAD10 protein to homogeneity (L. Bardwell, H. Burtscher, W. A. Weiss, C. M. Nicolet, and E. C. Friedberg, Biochemistry 29:3119-3126, 1990). In the present studies we show that the RAD1 protein, produced by in vitro transcription and translation of the cloned gene, specifically coimmunoprecipitates with the RAD10 protein translated in vitro or purified from yeast. Conversely, in vitro-translated RAD10 protein specifically coimmunoprecipitates with the RAD1 protein. The sites of this stable and specific interaction have been mapped to the C-terminal regions of both polypeptides. This portion of RAD10 protein is evolutionarily conserved. These results are the first biochemical evidence of a specific association between any eukaryotic proteins genetically identified as belonging to a recombination or DNA repair pathway and suggest that the RAD1 and RAD10 proteins act at the same or consecutive biochemical steps in both nucleotide excision repair and mitotic recombination.

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