Complementation of aprataxin deficiency by base excision repair enzymes

Çağlayan, Melike; Horton, Julie K.; Prasad, Rajendra; Wilson, Samuel H.

doi:10.1093/nar/gkv079

Cited by 31 publications

(70 citation statements)

References 28 publications

(43 reference statements)

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“…The DNA substrates with template bases A, T, C or G (A gap , T gap , C gap or G gap , Supplementary Table 1) were prepared as follows4950: The 5′-end FAM-labelled upstream oligonucleotide (19-mer) was annealed with the 3′-end FAM-labelled downstream oligonucleotide (17-mer) in the presence of the template oligonucleotide (37-mer), to generate a single-nucleotide gapped DNA substrate. Coupled BER assays including both pol β and Lig I or pol β and XRCC1/Lig III complex were conducted under steady-state conditions where the gapped DNA substrate was in excess (250 nM) over the enzyme mixture (100 nM).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, the adenylation reaction was performed in a reaction mixture (20 μl) containing 100 pmol oligonucleotide, 50 mM sodium acetate, pH 6.0, 10 mM MgCl 2 , 5 mM DTT, 0.1 mM EDTA and 0.1 mM ATP, and the reaction was initiated by adding 100 pmol Mth RNA ligase and incubated at 65 °C for 1 h. After heat inactivation of the enzyme at 85 °C for 5 min, the adenylated oligonucleotides were separated and then purified from other DNA species by electrophoresis in a 18% polyacrylamide gel containing 8 M urea in 89 mM Tris-HCl, 89 mM boric acid and 2 mM EDTA (pH 8.8). The DNA substrates with template bases A, T, C or G (A nick , T nick , C nick or G nick , Supplementary Table 1) were prepared as follows4950: the upstream oligonucleotide with 3′-8oxoG (18-mer) was annealed with the 3′-end FAM-labelled downstream oligonucleotide (16-mer) in the presence of the template oligonucleotide (34-mer) to generate the nicked DNA substrates. The ligation assays were performed in a reaction mixture (10 μl) containing 50 mM Tris(HCl), pH 7.5, 100 mM KCl, 10 mM MgCl 2 , 1 mM ATP, 100 μg ml −1 BSA, 10% glycerol, 1 mM DTT and 250 nM DNA substrate.…”

Section: Methodsmentioning

confidence: 99%

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Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

et al. 2017

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Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

et al. 2017

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“…For the reference reactions, start each reaction by adding the purified protein to the reaction mixture in final concentrations as follows: pol β (500 nM), APTX (100 nM), and FEN1 (100 nM) (Çağlayan et al ., 2014 and Çağlayan et al ., 2015). …”

Section: Methodsmentioning

confidence: 98%

“…The gel is scanned, and the data analyzed as reported (Çağlayan et al ., 2014 and Çağlayan et al ., 2015). …”

Section: Methodsmentioning

confidence: 98%

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Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells

Çağlayan¹,

Horton²,

Wilson³

2015

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We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts from Saccharomyces cerevisiae (Çağlayan and Wilson, 2014). Here, we describe a method to prepare cell extracts from vertebrate cells to investigate these enzymatic activities for the processing of the 5′-adenylated-sugar phosphate-containing BER intermediate. This new protocol complements our previous publication. The cell lines used are wild-type and APTX-deficient human lymphoblast cells from an Ataxia with Oculomotor Apraxia Type 1 (AOA1) disease patient, wild-type and APTX-null DT40 chicken B cells, and mouse embryonic fibroblast (MEF) cells. This protocol is a quick and efficient way to make vertebrate cell extracts without using commercial kits. Materials and Reagents 1.Cell lines used in this study a.The human cell lines used are the wild-type C2ABR and AOA1 L938 (Harris et al., 2009). The AOA1 cell line was derived from the peripheral blood of a Japanese AOA1 patient and has a point mutation within the HIT domain of APTX involving substitution of proline for leucine at position 206. b.The DT40 cell lines are the wild-type and APTX null. The APTX null cell line has the aptx gene deletion from valine 78 onwards that inactivates APTX (Ahel et al., 2006). c.The mouse embryonic fibroblast cell lines are pol β −/− and pol β +/+ MEFs previously developed in our laboratory (Sobol et al., 1996). 2.RPMI 1640 medium with glutamine (Gibco, catalog number: 11875-093) 3.DMEM high-glucose medium (HyClone, catalog number: SH30081) 4.Chicken serum (Life Technologies, catalog number 16110-082) 5.Glutamax-1 (Gibco, catalog number: 35050-061)

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Linking DNA polymerase theta structure and function in health and disease

Beagan

McVey

2015

Cell. Mol. Life Sci.

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DNA Polymerase theta (Pol θ) is an error-prone A-family polymerase that is highly conserved among multicellular eukaryotes and plays multiple roles in DNA repair and the regulation of genome integrity. Studies conducted in several model organisms have shown that Pol θ can be utilized during DNA interstrand crosslink repair and during alternative end-joining repair of double-strand breaks. Recent genetic and biochemical studies have begun to elucidate the unique structural features of Pol θ that promote alternative end-joining repair. Importantly, Pol θ-dependent end joining appears to be important for overall genome stability, as it affects chromosome translocation formation in murine and human cell lines. Pol θ has also been suggested to act as a modifier of replication timing in human cells, though the mechanism of action remains unknown. Pol θ is highly upregulated in a number of human cancer types, which could indicate that mutagenic Pol θ-dependent end joining is used during cancer cell proliferation. Here we review the various roles of Pol θ across species and discuss how these roles may be relevant to cancer therapy.

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Complementation of aprataxin deficiency by base excision repair enzymes

Cited by 31 publications

References 28 publications

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells

Linking DNA polymerase theta structure and function in health and disease

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