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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Main Authors: Jin Tang, Linlin Zhao
Format: Article
Language:English
Published: Elsevier 2022-01-01
Series:MethodsX
Subjects:
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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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