Apurinic/apyrimidinic endonuclease activities appear normal in the CHO-cell ethyl methanesulfonate-sensitive mutant, EM9

Belle, Michael La; Linn, Stuart; Thompson, Larry H.

doi:10.1016/0165-7992(84)90035-6

Cited by 27 publications

(16 citation statements)

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“…It is possible that there are other DNA ligase III abnormalities in EM9 that are not detected by the assay used in this work. For example, although the residual DNA ligase III activity in EM9 can apparently complete ligation events, as suggested by the observation that the adenylate-DNA ligase III intermediates are dissociated by the addition of oligo(dT) 16. poly(dA) (results not shown), it is possible that the ability of the enzyme to interact with other repair proteins is affected by the XRCC1 mutation.…”

Section: Discussionmentioning

confidence: 99%

“…This conclusion was confirmed by adding oligo(dT)16-poly(dA) or oligo(dT)16. poly(rA) to aliquots of preformed DNA ligase-adenylate intermediates to examine the substrate specificity of the IMAC-enriched DNA ligase activity. While only oligo(dT) 16. poly(dA) dissociated the adenylate group from the DNA ligase I control, both DNA substrates dissociated the adenylate group from the affinitypurified polypeptide (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Both of these cell lines rejoin DNA single-strand breaks arising from ionizing radiation and EMS at a reduced rate, suggesting a role for XRCC1 protein in the final stages of a base excision repair pathway. EM9 cell extracts were previously examined for abnormalities in several enzymes with putative roles in the repair of DNA strand breaks, all of which appeared normal (7,11,16). …”

Section: Discussionmentioning

confidence: 99%

“…Briefly, reaction mixtures contained 60 mM Tris-Cl (pH 8.0), 10 mM MgCl2, 5 mM dithiothreitol, 50 ,ug of BSA per ml, 1.5 or 10 ,uCi of [a-32P]ATP (3,000 Ci/mmol, 10 mCi/ml; Amersham), and indicated amounts of either purified calf thymus DNA ligase I (31) or extract from gel filtration or Ni-NTA chromatography. In experiments in which the substrate specificity of adenylylated polypeptides was examined, 30-,ul aliquots were removed from 150-pul reactions after 15 min, and either reactions were terminated as described above or the aliquots were incubated for a further 15 min alone or in the presence of 2 pug of one of the oligonucleotide substrates oligo(dT) 16. poly(dA) and oligo(dT)16. poly(rA) before termination.…”

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

et al. 1994

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The Chinese hamster ovary (CHO) cell mutant EM9 is hypersensitive to ethyl methanesulfonate (EMS) (10-fold) and ionizing radiation (1.8-fold), and it is unable to grow in medium containing chlorodeoxyuridine (CldUrd) under conditions in which 20% of genomic Thy is replaced by chlorouracil during DNA replication (9, 29). EM9 repairs -y-ray and EMS-induced single-strand breaks at a reduced rate and exhibits a 10-fold increase in the occurrence of sister chromatid exchange (SCE) (27,28). The sensitivity of EM9 to alkylating agents is suggestive of a defect in the base excision repair pathway, which involves sequential action by DNA glycosylase, apurinic-apyrimidinic endonuclease, deoxyribose-phosphodiesterase, DNA polymerase, and DNA ligase activities (17). The reduced rate of single-strand break rejoining suggests that the defect in EM9 lies within a postincision step of this pathway. EM9 is phenotypically similar to cells derived from individuals with Bloom's syndrome (BS), a cancer-prone autosomal recessive disorder characterized by high SCEs (10-fold) and sensitivity to alkylating agents (4,14,15 (31). As yet, no specific role has been identified for DNA ligase III (31). Altered DNA ligase activity in BS cells has been observed in several studies, in which it was proposed that the activity of DNA ligase I was affected (5, 6, 33, 34), but no major abnormality in DNA ligase activity was found in the one reported study in which EM9 was examined (7). However,

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Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

et al. 1994

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“…Because integration and expression of intact and linearized plasmid was normal in EM9 cells under a variety of conditions, our data imply that this defective aspect of homologous recombination may not be involved with integration or ligation. The relationship of this finding to the high sister chromatid exchange frequencies found in the mutant is unclear at this time, although it is known that the sister chromatid exchanges in EM9 appear to result mainly from the incorporated bromodeoxyuridine used in their detection (20) and that defects in DNA ligase (4), poly(ADP-ribose) metabolism (10), or apurinic/apyrimidimic endonuclease (13) are not involved. Furthermore, our results suggest that the enzymatic machinery by which mammalian cells process exogenous DNA overlaps with that which maintains genomic DNA.…”

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Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange.

1987

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Transformation frequencies were measured in CHO mutant EM9 after transfection with intact or modified plasmid pSV2-gpt. The mutant and wild-type strain behaved similarly under all conditions except when homologous recombination was required to produce an intact plasmid. Therefore, the defect of the mutant which renders it slow in DNA strand break rejoining and high in sister chromatid exchange induction reduces its ability to recombine foreign DNA molecules.Recombination of genomic sequences in mammalian cells is important in development and differentiation (25,26), the generation of sister chromatid exchange (14), and chromosomal translocations in cancer (41), among other important cellular processes. However, the mechanisms involved in recombination in mammalian cells have not been elucidated to the extent that they have been in procaryotes and simple eucaryotes (22,30). Recently, numerous studies have described systems for the study of recombination and ligation in mammalian cells. These systems generally involve the addition of foreign DNAs, such as animal viruses (38-40) or recombinant plasmids with selectable markers (5,6,17,21,27), and have shown that mammalian cells are able to efficiently reconstitute intact genes from gene fragments via homologous or nonhomologous recombination.In the present study, transformation to mycophenolic acid resistance of CHO mutant EM9 was measured after transfection with intact or modified pSV2-gpt, which codes for bacterial guanosine phosphoribosyltransferase (18,19), to assess integration, ligation, and recombination frequencies of the mutant compared with the wild-type strain, AA8 (36). Mutant EM9 has been characterized as having enhanced sensitivity to some, but not all, alkylating agents (9, 33, 37), slight sensitivity to X-irradiation (37), high base-line and induced sister chromatid exchange frequencies, and hampered ability to rejoin DNA strand breaks (33), making it an interesting candidate for the study of processing of exogenous DNA.A diagram of pSV2-gpt and relevant restriction sites is shown in Fig. 1A. For transfection experiments, DNA was calcium phosphate precipitated (35) and added to monolayers (2 x 106 cells per dish) of AA8 and EM9 cells obtained from suspension cultures grown in alpha-MEM (KC Biologicals) supplemented with 10% fetal bovine serum (Flow Laboratories, Inc.) (alpha-MEMFBS) as described previously (34). After DNA exposure, the cell monolayers were rinsed twice with phosphate-buffered saline and incubated in alpha-MEMFBS for 24 h. They were then transferred to roller tubes for suspension growth. After the appropriate time for expression of the Gpt phenotype, cells were counted and plated for plating efficiency measurements in alpha-MEMFBS (300 cells per dish) or for selection (105 to 106 cells per dish) in MAXTA medium (alpha-MEMFBS

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A review on protein–protein interaction network of APE1/Ref‐1 and its associated biological functions

Thakur

Dhiman

Tell

et al. 2015

Cell Biochemistry & Function

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Apurinic/apyrimidinic endonuclease 1 (APE1) is a classic example of functionally variable protein. Besides its well-known role in (i) DNA repair of oxidative base damage, APE1 also plays a critical role in (ii) redox regulation of transcription factors controlling gene expression for cell survival pathways, for which it is also known as redox effector factor 1 (Ref-1), and recent evidences advocates for (iii) coordinated control of other non-canonical protein-protein interaction(s) responsible for significant biological functions in mammalian cells. The diverse functions of APE1 can be ascribed to its ability to interact with different protein partners, owing to the attainment of unfolded domains during evolution. Association of dysregulation of APE1 with various human pathologies, such as cancer, cardiovascular diseases and neurodegeneration, is attributable to its multifunctional nature, and this makes APE1 a potential therapeutic target. This review covers the important aspects of APE1 in terms of its significant protein-protein interaction(s), and this knowledge is required to understand the onset and development of human pathologies and to design or improve the strategies to target such interactions for treatment and management of various human diseases.

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Apurinic/apyrimidinic endonuclease activities appear normal in the CHO-cell ethyl methanesulfonate-sensitive mutant, EM9

Cited by 27 publications

References 16 publications

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

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

Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange.

A review on protein–protein interaction network of APE1/Ref‐1 and its associated biological functions

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