Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites
DNA interstrand cross-links (ICLs) are extremely deleterious DNA lesions, which can block different DNA transactions. A major step in ICL repair involves strand cleavage activities flanking the cross-linking site, also known as unhooking. The cleavage generates a single-stranded DNA remnant attached...
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Elsevier
2022-01-01
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Online Access: | http://www.sciencedirect.com/science/article/pii/S2215016122000711 |
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author | Jin Tang Linlin Zhao |
author_facet | Jin Tang Linlin Zhao |
author_sort | Jin Tang |
collection | DOAJ |
description | DNA interstrand cross-links (ICLs) are extremely deleterious DNA lesions, which can block different DNA transactions. A major step in ICL repair involves strand cleavage activities flanking the cross-linking site, also known as unhooking. The cleavage generates a single-stranded DNA remnant attached to the unbroken strand, often referred to as the unhooked ICL repair intermediates. The unhooked ICLs are substrates for specialized DNA polymerases, leading to the eventual restoration of the duplex DNA structure. Although these repair events have been outlined, the understanding of molecular details of the repair pathways has been hindered by the difficulty of preparing structurally defined ICL repair intermediates. Here, we present a straightforward method to prepare model ICL repair intermediates derived from a ubiquitous type of endogenous DNA modification, abasic (AP) sites. AP-derived ICLs have emerged as an important type of endogenous ICLs. We developed the method based on commercially available materials without the requirement of synthetic chemistry expertise. The method is expected to be accessible to any interested labs in the DNA repair community.• The method exploits the alkaline lability of ribonucleotides and uses designer oligonucleotides to create ICL repair intermediates with varying lengths of the unhooked strand.• Strand cleavage at ribonucleotides is achieved using NaOH, which avoids the potential for incomplete digestion during enzymatic workup due to specific substrate structures.• The method is grounded on the high cross-linking yield between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination, developed by Gates and colleagues. |
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institution | Directory Open Access Journal |
issn | 2215-0161 |
language | English |
last_indexed | 2024-04-13T05:28:07Z |
publishDate | 2022-01-01 |
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series | MethodsX |
spelling | doaj.art-082a7e80ae4743e195d98d314890320c2022-12-22T03:00:32ZengElsevierMethodsX2215-01612022-01-019101687Preparation of DNA interstrand cross-link repair intermediates induced by abasic sitesJin Tang0Linlin Zhao1University of California, Riverside, CA, USACorresponding author.; University of California, Riverside, CA, USADNA interstrand cross-links (ICLs) are extremely deleterious DNA lesions, which can block different DNA transactions. A major step in ICL repair involves strand cleavage activities flanking the cross-linking site, also known as unhooking. The cleavage generates a single-stranded DNA remnant attached to the unbroken strand, often referred to as the unhooked ICL repair intermediates. The unhooked ICLs are substrates for specialized DNA polymerases, leading to the eventual restoration of the duplex DNA structure. Although these repair events have been outlined, the understanding of molecular details of the repair pathways has been hindered by the difficulty of preparing structurally defined ICL repair intermediates. Here, we present a straightforward method to prepare model ICL repair intermediates derived from a ubiquitous type of endogenous DNA modification, abasic (AP) sites. AP-derived ICLs have emerged as an important type of endogenous ICLs. We developed the method based on commercially available materials without the requirement of synthetic chemistry expertise. The method is expected to be accessible to any interested labs in the DNA repair community.• The method exploits the alkaline lability of ribonucleotides and uses designer oligonucleotides to create ICL repair intermediates with varying lengths of the unhooked strand.• Strand cleavage at ribonucleotides is achieved using NaOH, which avoids the potential for incomplete digestion during enzymatic workup due to specific substrate structures.• The method is grounded on the high cross-linking yield between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination, developed by Gates and colleagues.http://www.sciencedirect.com/science/article/pii/S2215016122000711Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites |
spellingShingle | Jin Tang Linlin Zhao Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites MethodsX Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites |
title | Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites |
title_full | Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites |
title_fullStr | Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites |
title_full_unstemmed | Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites |
title_short | Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites |
title_sort | preparation of dna interstrand cross link repair intermediates induced by abasic sites |
topic | Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites |
url | http://www.sciencedirect.com/science/article/pii/S2215016122000711 |
work_keys_str_mv | AT jintang preparationofdnainterstrandcrosslinkrepairintermediatesinducedbyabasicsites AT linlinzhao preparationofdnainterstrandcrosslinkrepairintermediatesinducedbyabasicsites |