A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

Boorstein, Robert J.; Chiu, Lai-No; Teebor, George W.

doi:10.1128/mcb.12.12.5536

Cited by 49 publications

(39 citation statements)

References 29 publications

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“…When cells were co-exposed to hmdUrd and 4-AN for 24 h, sensitization was not observed (data not shown). This "scheduling requirement" is consistent with the idea that hmdUrd has first to be incorporated into cellular DNA and that cytotoxicity is not observed until the unnatural base is removed by glycosylase (SMUG1)-initiated BER (25).…”

Section: -An-induced Sensitization To Mms Is S-phase-dependent-supporting

confidence: 82%

Poly(ADP-ribose) Polymerase Activity Prevents Signaling Pathways for Cell Cycle Arrest after DNA Methylating Agent Exposure

Horton

Stefanick

Naron

et al. 2005

Journal of Biological Chemistry

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Mouse fibroblasts, deficient in DNA polymerase ␤, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase ␤ null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G 2 /M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.Removal of unnatural bases or single base lesions from DNA is predominantly by a glycosylase-initiated base excision repair (BER) 1 pathway. Methylated bases are excised by N-methylpurine-DNA glycosylase, a monofunctional glycosylase. In the preferred "single-nucleotide" BER pathway, this is followed by strand cleavage on the 5Ј side of the sugar by apurinic/apyrimidinic endonuclease, gap filling and cleavage on the 3Ј side by the DNA synthesis and deoxyribose phosphate (dRP) lyase activities, respectively, of DNA polymerase ␤ (␤-pol), and finally sealing of the nick by a DNA ligase (1). The essential role of ␤-pol in this pathway has been established using extracts from wild-type and ␤-pol null mouse fibroblasts (2). The requirement for ␤-pol, more specifically the dRP lyase activity of ␤-pol, in protection of cells against the cytotoxicity of methyl DNA lesions, has been demonstrated by the hypersensitivity of ␤-pol null cells to the monofunctional methylating agent methyl methanesulfonate (MMS) (2, 3). It is thought that the MMS hypersensitivity phenotype of ␤-pol null cells reflects accumulation of cytotoxic repair intermediates, such as the 5Ј dRP group after removal of methylated bases from DNA (4).Repair of oxidative DNA damage occurs by an alternate sub-pathway of single-nucleotide BER utilizing bifunctional glycosylases that have an associated lyase activity that nicks the DNA strand 3Ј to the abasic site after base removal. Singlen...

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Section: -An-induced Sensitization To Mms Is S-phase-dependent-supporting

confidence: 82%

Poly(ADP-ribose) Polymerase Activity Prevents Signaling Pathways for Cell Cycle Arrest after DNA Methylating Agent Exposure

Horton

Stefanick

Naron

et al. 2005

Journal of Biological Chemistry

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“…HmdU can be incorporated into the DNA of living cells and triggers cell death when HmdU incorporation exceeds a threshold (17)(18)(19). The incorporation of exogenous HmdU can provide a method of introducing into the DNA a single form of DNA damage repaired by the base excision repair pathway.…”

Section: Discussionmentioning

confidence: 99%

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Rogstad

Darwanto

Herring

et al. 2007

Chem. Res. Toxicol.

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The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage. More recently, the corresponding deoxynucleoside, 5-hydroxymethyl-2′-deoxyuridine (HmdU) has proven to be valuable for the introduction of controlled amounts of a single type of damage lesion into the DNA of replicating cells, which is subsequently repaired by the base excision repair pathway. Complicating the study of HmU formation and repair, however, is the known chemical reactivity of the hydroxymethyl group of HmU under conditions used to hydrolyze DNA. In the work reported here, this chemical property has been exploited by creating conditions that convert HmU to the corresponding methoxymethyluracil (MmU) derivative that can be further derivatized to the 3,5-bis-(trifluoromethyl)benzyl analog. This derivatized compound can be detected by gas chromatographynegative chemical ionization-mass spectrometry (GC-NCI-MS) with good sensitivity. Using isotopically enriched exogenous HmdU and human osteosarcoma cells (U2OS) in culture, we demonstrate that this method allows for the measurement of HmU in DNA formed from incorporation of exogenous HmdU. We further demonstrate that addition of isotopically enriched uridine to the culture medium allows for the simultaneous measurement of DNA replication and repair kinetics. This sensitive and facile method should prove valuable for studies on DNA oxidation damage and repair in living cells.

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“…In support of this conclusion, pol β-deficient cells are also hypersensitive to the thymidine analog, 5-hydroxymethyl-2′-deoxyuridine (hmdUrd) [24]. This agent is incorporated into cellular DNA and elicits cytotoxicity only once removed by glycosylaseinitiated BER [29].…”

Section: Mammalian Base Excision Repair (Ber)mentioning

confidence: 94%

Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency

Horton¹,

Wilson²

2007

DNA Repair

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Single-base lesions in DNA are repaired predominantly by base excision repair (BER). DNA polymerase β (pol β) is the polymerase of choice in the preferred single-nucleotide BER pathway. The characteristic phenotype of mouse fibroblasts with a deletion of the pol β gene is moderate hypersensitivity to monofunctional alkylating agents, e.g., methyl methanesulfonate (MMS). Increased sensitivity to MMS is also seen in the absence of pol β partner proteins XRCC1 and PARP-1, and under conditions where BER efficiency is reduced by synthetic inhibitors. PARP activity plays a major role in protection against MMS-induced cytotoxicity, and cells treated with a combination of non-toxic concentrations of MMS and a PARP inhibitor undergo cell cycle arrest and die by a Chk1-dependent apoptotic pathway. Since BER-deficient cells and tumors are similarly hypersensitive to the clinically used chemotherapeutic methylating agent temozolomide, modulation of DNA damage-induced cell signaling pathways, as well as BER, are attractive targets for potentiating chemotherapy.

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A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

Cited by 49 publications

References 29 publications

Poly(ADP-ribose) Polymerase Activity Prevents Signaling Pathways for Cell Cycle Arrest after DNA Methylating Agent Exposure

Poly(ADP-ribose) Polymerase Activity Prevents Signaling Pathways for Cell Cycle Arrest after DNA Methylating Agent Exposure

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency

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