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. 2022 Jun 3;10:826576. doi: 10.3389/fcell.2022.826576

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Copyright © 2022 Biswas, Zhao, Makinwa, Bassett, Musich, Liu and Zou.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Effect of cis/trans isomerization on ATR oligomerization and ATR–ATRIP complex formation. (A) Oligomerization of trans- and cis-ATR was analyzed by co-expressing vector constructs encoding FLAG-ATR, Myc-ATR, and HA-ATRIP transfected into the ATR^flox/− cells followed by UV+/− treatments. ATR oligomerization was assessed by co-immunoprecipitation after cellular fractionation. Interestingly, no ATRIP occurred in the cytoplasm as compared to ATRIP in the nucleus. The Myc/FLAG ratios were normalized to the ratio at lane 11 (cytoplasm fraction) and HA/FLAG at lane 11 (Nuclear fraction). (B) PLA revealing a direct interaction between FLAG-ATR and Myc-ATR in ATR^flox/− cells co-transfected with FLAG-ATR and Myc-ATR plasmids. The interaction was enhanced after UV irradiation at 40 J/m². A DAPI-staining overlay shows the location of the nuclei. (C) ATR/ATRIP complex formation was monitored using PLA by co-transfecting FLAG-ATR WT, P429A, or S428A mutant with Myc-ATRIP in ATR^flox/- cells. (D) ATRIP oligomerization in the presence of FLAG-ATR WT, P429A, or S428A mutant proteins. ATR^flox/- cells were co-transfected with Myc- and HA-ATRIP plus FLAG-ATR WT, P429A, or S428A constructs followed by UV irradiation. Oligomerization of Myc- and HA-ATRIP was monitored by PLA. Foci formation per cells was calculated considering an average of 50 cells, and each experiment was performed in triplicate. The bar graphs represent the statistical analysis of the PLA images. * stands for the p-value < 0.01.