@article{71b70f742c98486cb3ea824952f76474,
title = "EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart",
abstract = "Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.",
keywords = "ATR phosphorylation, Bloom, Fanconi anemia, SCE, exonuclease, fork restart, genetic instability, interstrand crosslink repair, replication stress, sister chromatid exchange, tumor proliferation",
author = "Shashank Hambarde and Tsai, {Chi Lin} and Pandita, {Raj K.} and Albino Bacolla and Anirban Maitra and Vijay Charaka and Hunt, {Clayton R.} and Rakesh Kumar and Oliver Limbo and {Le Meur}, Remy and Chazin, {Walter J.} and Tsutakawa, {Susan E.} and Paul Russell and Katharina Schlacher and Pandita, {Tej K.} and Tainer, {John A.}",
note = "Funding Information: We thank Arnab R. Chaudhuri, Susan Lees-Miller, Chris Brosey, Aleem Syed, Zu Ye, and Kalpana Mujoo for input and Xiaoyan Wang and Naga Babu Chinnam for testing full-length EXO5 stability. T.K.P. received WT GFP-EXO5 plasmid from Peter M. Burgers and EXO5 KO LNCaP cells from Binghui Shen. J.A.T. is supported by NIH grants ( P01 CA092584 , R35CA220430 ), a Robert A. Welch Chemistry Chair , and Cancer Prevention and Research Institute of Texas (CPRIT) grant RP180813 . T.K.P. is supported by NIH grants RO1 CA129537 and RO1 GM109768 and the Houston Methodist Research Institute . K.S. is supported by National Institute of Environmental Health Sciences (NIEHS) grant 1R01ES029680 and CPRIT grants RP180463 and RP180813 . K.S. is a Rita Allen Foundation Fellow and a CPRIT scholar in cancer biology. TCGA research used the Texas Advanced Computing Center, supported by National Science Foundation (NSF) grant ACI-1134872 . X-ray diffraction data were collected at SSRL beamline 12-2 and ALS beamlines 12.3.1 (supported by NIH project ALS-ENABLE [ P30 GM124169 ] and the Integrated Diffraction Analysis Technologies [IDAT] program) and 8.3.1 (supported by NIH grants R01 GM124149 and P30 GM124169 ). Funding Information: We thank Arnab R. Chaudhuri, Susan Lees-Miller, Chris Brosey, Aleem Syed, Zu Ye, and Kalpana Mujoo for input and Xiaoyan Wang and Naga Babu Chinnam for testing full-length EXO5 stability. T.K.P. received WT GFP-EXO5 plasmid from Peter M. Burgers and EXO5 KO LNCaP cells from Binghui Shen. J.A.T. is supported by NIH grants (P01 CA092584, R35CA220430), a Robert A. Welch Chemistry Chair, and Cancer Prevention and Research Institute of Texas (CPRIT) grant RP180813. T.K.P. is supported by NIH grants RO1 CA129537 and RO1 GM109768 and the Houston Methodist Research Institute. K.S. is supported by National Institute of Environmental Health Sciences (NIEHS) grant 1R01ES029680 and CPRIT grants RP180463 and RP180813. K.S. is a Rita Allen Foundation Fellow and a CPRIT scholar in cancer biology. TCGA research used the Texas Advanced Computing Center, supported by National Science Foundation (NSF) grant ACI-1134872. X-ray diffraction data were collected at SSRL beamline 12-2 and ALS beamlines 12.3.1 (supported by NIH project ALS-ENABLE [P30 GM124169] and the Integrated Diffraction Analysis Technologies [IDAT] program) and 8.3.1 (supported by NIH grants R01 GM124149 and P30 GM124169). J.A.T. and T.K.P. directed the study. C.-L.T. S.H. A.B. J.A.T. and T.K.P. contributed to design. C.-L.T. did structural, biochemical, and in vitro binding experiments. A.B. did bioinformatic analyses. S.H. R.K.P. V.C. C.R.H. and R.K. did cellular experiments. K.S. examined DNA fiber analyses. R.M. and W.J.C. provided RPA. O.L. and P.R. did fission yeast genetic experiments. C.-L.T. S.H. A.B. C.R.H. S.E.T. K.S. T.K.P. and J.A.T. wrote the manuscript. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2021 Elsevier Inc.",
year = "2021",
month = jul,
day = "15",
doi = "10.1016/j.molcel.2021.05.027",
language = "English (US)",
volume = "81",
pages = "2989--3006.e9",
journal = "Molecular cell",
issn = "1097-2765",
publisher = "Cell Press",
number = "14",
}