Abstract
The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. The APTX RNA‐DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. Here, we report X‐ray structures of APTX engaging nicked RNA‐DNA substrates that provide direct evidence for a wedge‐pivot‐cut strategy for 5′‐AMP resolution shared with the alternate 5′‐AMP processing enzymes POLβ and FEN1. Our results uncover a DNA‐induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Further, based on comprehensive biochemical, X‐ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations.
Highlights
The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks
The structural snapshots captured in our crystal structures are representative of APTX bound to a nicked RNA-50-30-DNA junction reaction product complex formed by APTX during repair of abortive ligation products created during ribonucleotide excision repair (RER) (Tumbale et al, 2014; Schellenberg et al, 2015)
Global conformational responses of the APTX catalytic domain during catalysis To better define the response of the APTX catalytic domain (APTXcat) to substrate binding in solution, we investigated the behavior of 13CH3-Met-labeled APTXcat by nuclear magnetic resonance (NMR)
Summary
The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. We report X-ray structures of APTX engaging nicked RNA-DNA substrates that provide direct evidence for a wedge-pivot-cut strategy for 50-AMP resolution shared with the alternate 50-AMP processing enzymes POLb and FEN1. Our results uncover a DNA-induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Based on comprehensive biochemical, X-ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations
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