Rapid and Efficient DNA Strand Cross‐Linking by Click Chemistry

Kočalka, Petr; El‐Sagheer, Afaf H.; Brown, Tom

doi:10.1002/cbic.200800006

Cited by 83 publications

(81 citation statements)

References 43 publications

(34 reference statements)

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“…The substrates contain a single site‐specific triazole ICL placed at the fork junction (Figs 4A and EV5, Appendix Fig S1; Kocalka et al , 2008). We analysed the activity of XPF‐ERCC1 on this substrate in the presence and absence of RPA and nascent model leading strand, initially employing 3′‐end‐labelled substrates.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to the differences in ICL location and the enzymology employed (here, RPA and SNM1A were also considered), the structure of the substrates used in the study of Kuraoka et al and those presented here are substantially different. The ICLs we have used produce relatively little distortion of the DNA (Kocalka et al , 2008), in contrast to the psoralen ICLs used by Kuraoka et al Together with the location of the ICL relative to the fork junction, this could also have a substantial impact on the position and efficiency of incisions.…”

Section: Discussionmentioning

confidence: 97%

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RPA activates the XPF ‐ ERCC 1 endonuclease to initiate processing of DNA interstrand crosslinks

et al. 2017

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During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 97%

RPA activates the XPF ‐ ERCC 1 endonuclease to initiate processing of DNA interstrand crosslinks

et al. 2017

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“…Azides are unstable to P III , and do not survive the conditions of phosphoramidite oligonucleotide synthesis. Therefore, the azide functionalities were introduced after oligonucleotide assembly by selective reaction of the N-hydroxysuccinimide ester of azidohexanoic acid 2 with the primary amino groups of the modified uracil bases (26). All oligonucleotides were purified by reversed-phase HPLC and analyzed by mass spectrometry prior to click ligation.…”

Section: Resultsmentioning

confidence: 99%

New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes

El‐Sagheer

Brown

2010

Proc. Natl. Acad. Sci. U.S.A.

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The CuAAC reaction (click chemistry) has been used in conjunction with solid-phase synthesis to produce catalytically active hairpin ribozymes around 100 nucleotides in length. Cross-strand ligation through neighboring nucleobases was successful in covalently linking presynthesized RNA strands with high efficiency (transligation). In an alternative strategy, intrastrand click ligation was employed to produce a functional hammerhead ribozyme containing a novel nucleic acid backbone mimic at the catalytic site (cisligation). The ability to synthesize long RNA strands by a combination of solid-phase synthesis and click ligation is an important addition to RNA chemistry. It is compatible with a plethora of sitespecific modifications and is applicable to the synthesis of many biologically important RNA molecules.ligonucleotide chemistry is central to the advancement of core technologies such as DNA sequencing and genetic analysis and has impacted greatly on the discipline of molecular biology (1, 2). Oligonucleotides and their analogues are essential tools in these areas. They are produced by automated solid-phase phosphoramidite synthesis, a highly efficient process that can be used to assemble DNA strands over 100 bases in length (3). Synthesis of RNA is less efficient owing to problems caused by the presence of the 2′-hydroxyl group of ribose, which requires selective protection during oligonucleotide assembly. This reduces the coupling efficiency of RNA phosphoramidite monomers due to steric hindrance. In addition, side-reactions that occur during the removal (or premature loss) of the 2′-protecting groups cause phosphodiester backbone cleavage and 3′ to 2′ phosphate migration (4). Although several ingenious strategies have been developed to minimize these problems (5) and to improve the synthesis of long RNA (6), the chemical complexity of solid-phase RNA synthesis dictates that constructs longer than 50 nucleotides in length remain difficult to prepare. Most biologically important RNA molecules such as ribozymes (7), aptamers (8), and riboswitches (9, 10) are significantly longer than this, so new approaches to the synthesis of RNA analogues are urgently required. Although RNA synthesis by transcription might seem a viable alternative, it does not permit the site-specific incorporation of multiple modifications at sugars, bases, or phosphates. In contrast, automated solid-phase RNA synthesis is compatible with the introduction of fluorescent tags, isotopic labels (for NMR studies) and other groups to improve biological activity and resistance to enzymatic degradation. The scope and utility of important RNA constructs can be significantly extended by such chemical modifications, and the scale of chemical synthesis is potentially unlimited (11). These are important factors when planning structural studies on RNA or the development of therapeutic oligoribonucleotides analogues, and should be considered when devising new methods of RNA assembly. Enzymatic ligation might appear to be a method of achieving some of th...

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“…41 For this purpose a single deoxyuridine (dU) nucleoside modified with a terminal alkyne was incorporated into a 14-mer DNA strand during solid-phase synthesis using either 5-ethynyl or 5-(octa-1,7-diynyl)-2 0 -deoxyuridine phosphoramidite building blocks 1b or 2b (Fig. 8).…”

Section: Oligonucleotide Cross-linkingmentioning

confidence: 99%

“…41 In a study to develop methods of attaching oligonucleotides to surfaces, solid phase non-templated chemical ligation of pairs of oligonucleotides by the CuAAC reaction has been used to produce oligonucleotides joined by 5 0 -5 0 linkages (Scheme 11). 56 Such ODNs are of potential value in the triplex field.…”

Section: Oligonucleotide Ligationmentioning

confidence: 99%