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bioRxiv · 10.1101/2021.05.11.443618

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

Le Meur, R. A.·Pecen, T. J.·Le Meur, K. V.·Nagel, Z. D.·Chazin, W. J.
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Abstract

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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Le Meur, R. A., Pecen, T. J., Le Meur, K. V., Nagel, Z. D., Chazin, W. J.. 2021-05-11. Molecular basis and functional consequences of the interaction between the Base Excision Repair DNA glycosylase NEIL1 and RPA. https://doi.org/10.1101/2021.05.11.443618

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