A key interaction with RPA orients XPA in NER complexes

Topolska-Woś, Agnieszka M.; Sugitani, Norie; Cordoba, John J; Meur, Kateryna V Le; Meur, Rémy Le; Kim, Hyun Suk; Yeo, Jung-Eun; Rosenberg, Daniel; Hammel, Michal; Schärer, Orlando D.; Chazin, Walter J.

doi:10.1093/nar/gkz1231

Cited by 46 publications

(76 citation statements)

References 63 publications

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“…RPA physically interacts with over three dozen DNA processing enzymes and recruits several of them onto DNA ( 4 ). Finally, RPA hands-off the DNA to these enzymes and correctly positions them to facilitate their respective catalytic activity ( 5 ). Plasticity for such multiplexed functional roles for RPA can be ascribed to its flexible structure and its corresponding context-dependent interactions with DNA ( 6 , 7 ).…”

Section: Introductionmentioning

confidence: 99%

Hydrogen–deuterium exchange reveals a dynamic DNA-binding map of replication protein A

Ahmad

Patterson

Deveryshetty

et al. 2021

Nucleic Acids Research

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Replication protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes. RPA binds ssDNA utilizing multiple oligosaccharide/oligonucleotide binding domains and based on their individual DNA binding affinities are classified as high versus low-affinity DNA-binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen–deuterium exchange mass spectrometry (HDX-MS), we assessed the ssDNA-driven dynamics of the individual domains of human RPA. As expected, ssDNA binding shows HDX changes in DBDs A, B, C, D and E. However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. Slower allosteric changes transpire in the protein–protein interaction domains and linker regions, and thus do not directly interact with ssDNA. Within a dynamics-based model for RPA, we propose that DBD-A and -B act as the dynamic half and DBD-C, -D and -E function as the less-dynamic half. Thus, segments of ssDNA buried under the dynamic half are likely more readily accessible to RPA-interacting proteins.

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Section: Introductionmentioning

confidence: 99%

Hydrogen–deuterium exchange reveals a dynamic DNA-binding map of replication protein A

Ahmad

Patterson

Deveryshetty

et al. 2021

Nucleic Acids Research

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“…Such movement may represent a transition state on the path to a stable “unwound” state. In our samples, however, such transition may have been present only transiently, easily reverting back to the “pre-unwound” state; complete and stable conversion into the “unwound” state may require additional factors such as Rad14(XPA) and RPA 83 – 85 accompanied by the large movement of Rad3/XPD as described above 43 . Also, a longer DNA segment on the 5' side of the lesion than our current design may be required in order to permit a larger bubble formation.…”

Section: Discussionmentioning

confidence: 86%

Cryo-EM structure of TFIIH/Rad4–Rad23–Rad33 in damaged DNA opening in nucleotide excision repair

et al. 2021

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The versatile nucleotide excision repair (NER) pathway initiates as the XPC–RAD23B–CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4–Rad23-Rad33 (yeast homologue of XPC–RAD23B–CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9–9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.

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“…In previous studies we often observed that the interaction of RPA with partner proteins have, in addition to a readily characterized interaction with RPA70N or RPA32C, a secondary interaction with the tandem high affinity DNA binding domains RPA70AB (e.g. (27)). These interactions are invariably weaker than the contacts with the corresponding protein recruitment domain.…”

Section: Resultsmentioning

confidence: 99%

Molecular basis and functional consequences of the interaction between the Base Excision Repair DNA glycosylase NEIL1 and RPA

Meur

et al. 2021

Preprint

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NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein replication protein A (RPA) modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ~ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against ionizing radiation-induced DNA lesions in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed that RPA interaction is not required for NEIL1 activity against oxidative damage in duplex DNA, and furthermore revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.

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A key interaction with RPA orients XPA in NER complexes

Cited by 46 publications

References 63 publications

Hydrogen–deuterium exchange reveals a dynamic DNA-binding map of replication protein A

Hydrogen–deuterium exchange reveals a dynamic DNA-binding map of replication protein A

Cryo-EM structure of TFIIH/Rad4–Rad23–Rad33 in damaged DNA opening in nucleotide excision repair

Molecular basis and functional consequences of the interaction between the Base Excision Repair DNA glycosylase NEIL1 and RPA

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