Characterization of DNA-protein crosslinks formed by treatment of L1210 cells and nuclei with bis(2-chloroethyl)methylamine (nitrogen mustard)

Thomas, Cornelius B.; Kohn, Kurt W.; Bonner, William M.

doi:10.1021/bi00612a012

Cited by 27 publications

(24 citation statements)

References 8 publications

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“…The resultant monoadduct can react with water to terminate the reaction, or there may be a second reaction with guanine (or adenine (Balcome et al , 2004)) on the opposite strand to form an interstrand DNA crosslink. Reaction with cysteines in chromosomal proteins, producing protein DNA crosslinks is also possible (Thomas et al , 1978; Loeber et al , 2009). The DNA monoadduct is the dominant product (90–95%), while of the ICLs (~5%) the N7 to N7 guanine-guanine diadduct is the major component.…”

Section: Interstrand Dna Crosslinking Agentsmentioning

confidence: 99%

DNA interstrand crosslink repair in mammalian cells: step by step

Muniandy

Liu

Majumdar

et al. 2009

Critical Reviews in Biochemistry and Molecular Biology

163

168

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Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells.

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Section: Interstrand Dna Crosslinking Agentsmentioning

confidence: 99%

DNA interstrand crosslink repair in mammalian cells: step by step

Muniandy

Liu

Majumdar

et al. 2009

Critical Reviews in Biochemistry and Molecular Biology

163

168

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“…11–14 In contrast, little is known about the composition and cellular abundance of the corresponding DNA-protein lesions. 15–17 …”

Section: Introductionmentioning

confidence: 99%

Mechlorethamine-Induced DNA–Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

et al. 2011

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Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins which were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI+-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.

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“…DPCs can form endogenously a result of oxidative stress and lipid peroxidation,2,3 or can be induced by exposure to ionizing radiation,4 metals,5 or common chemotherapeutic agents such as nitrogen mustards,6,7 platinum drugs,8 and alkylnitrosoureas 9. Although their biological relevance is poorly understood, certain types of DPCs persist through several cycles of DNA replication,1,10 potentially leading to cytotoxic and mutagenic outcomes such as sister chromatid exchanges and large deletions.…”

Section: Introductionmentioning

confidence: 99%

DNA−Protein Cross-Linking by 1,2,3,4-Diepoxybutane

et al. 2010

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Abstract1,2,3,4-Diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate carcinogenic metabolite of the common industrial chemical and environmental carcinogen 1,3-butadiene. DEB is a bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs), which are thought to play a central role in its biological activity. Previous studies with recombinant proteins have shown that the biological outcomes of DEB-induced DPCs are strongly influenced by protein identities. The present work combines affinity capture methodology with mass spectrometry-based proteomics and immunological detection to identify the proteins which form DPCs in nuclear extracts from human cervical carcinoma (HeLa) cells. We identified 39 human proteins that form covalent DPCs in the presence of DEB. DNA-protein cross-linking efficiency following treatment with 25 mM DEB was 2-12%, depending on protein identity. HPLC-ESI + -MS/MS analysis of the total proteolytic digests of cross-linked proteins revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, suggesting that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA.

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Characterization of DNA-protein crosslinks formed by treatment of L1210 cells and nuclei with bis(2-chloroethyl)methylamine (nitrogen mustard)

Cited by 27 publications

References 8 publications

DNA interstrand crosslink repair in mammalian cells: step by step

DNA interstrand crosslink repair in mammalian cells: step by step

Mechlorethamine-Induced DNA–Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

DNA−Protein Cross-Linking by 1,2,3,4-Diepoxybutane

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