The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3′-5′ nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3′-5′ exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.oxidative stress | APE2 | Zf-GRF | crystallography | Xenopus laevis

Highlights d Maelstrom represses canonical transcription within fly dualstrand piRNA clusters d Maelstrom also represses canonical transcription in conventional heterochromatin d Maelstrom can be guided to its targets both by piRNAs and piRNA-independent mechanisms

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dualstrand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the Heterochromatin Protein 1 homolog, Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. In addition to Mael, the piRNA biogenesis factors Armitage and Piwi, but not Rhi, are required to repress canonical transcription in dual-strand clusters. We propose that Armitage, Piwi, and Mael collaborate to repress potentially dangerous transcription of individual transposon mRNAs within clusters, while Rhi allows noncanonical transcription of the clusters into piRNA precursors without generating transposase-encoding mRNAs.

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