POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells

TY - JOUR

T1 - POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells

AU - Belan, Ondrej

AU - Sebald, Marie

AU - Adamowicz, Marek

AU - Anand, Roopesh

AU - Vancevska, Aleksandra

AU - Neves, Joana

AU - Grinkevich, Vera

AU - Hewitt, Graeme

AU - Segura-Bayona, Sandra

AU - Bellelli, Roberto

AU - Robinson, Helen M R

AU - Higgins, Geoff S

AU - Smith, Graeme C M

AU - West, Stephen C

AU - Rueda, David S

AU - Boulton, Simon J

N1 - Funding Information: Work in the Boulton lab is supported by the Francis Crick Institute (cc2098), European Research Council Advanced Investigator (TelMetab), Wellcome Trust Senior Investigator grants, and CRUK RadNet City of London. The Single Molecule Imaging Group is funded by a core grant of the MRC-London Institute of Medical Sciences (UKRI MC-A658-5TY10). S.J.B. and D.S.R. are also funded by a Wellcome Trust Collaborative Grant (P67153). Work in the West group is supported by the Francis Crick Institute (cc2098), BBSRC (BB/W01355X/1), and the Louis-Jeantet Foundation. Work in the Higgins lab is funded by the CRUK RadNet Oxford and CRUK Clinician Scientist funding (C34326/A19590). S.S.-B. was the recipient of an EMBO LT Fellowship (ALTF 707-2019) and an MSCA-IF (886577). S.J.B. and O.B. conceived the study. A.V. made comments. O.B. and R.A. expressed, purified, and fluorescently labeled the proteins and performed single-molecule and ensemble assays; O.B. M.S. M.A. and A.V. performed a subset of cell-based assays and analyzed the data. V.G. J.N. G.S.H. and G.C.M.S. generated SUM149PT-derived cell lines and performed a subset of assays. S.S.-B. and G.H. generated POLQ KO cells. O.B. and S.J.B. wrote the manuscript with editorial help from R.A. M.A. M.S. R.B. and D.S.R. S.J.B is a co-founder, VP Science Strategy and a shareholder of Artios Pharma Ltd. G.S.H. received consultancy fees from and is a shareholder of Artios Pharma Ltd and is a member of the Molecular Cell advisory board. V.G. J.N. H.M.R.R. and G.C.M.S. are all employees and shareholders of Artios Pharma Ltd. G.C.M.S. is a shareholder of AstraZeneca PLC. Funding Information: Work in the Boulton lab is supported by the Francis Crick Institute ( cc2098 ), European Research Council Advanced Investigator (TelMetab), Wellcome Trust Senior Investigator grants , and CRUK RadNet City of London . The Single Molecule Imaging Group is funded by a core grant of the MRC-London Institute of Medical Sciences ( UKRI MC-A658-5TY10 ). S.J.B. and D.S.R. are also funded by a Wellcome Trust Collaborative Grant ( P67153 ). Work in the West group is supported by the Francis Crick Institute ( cc2098 ), BBSRC ( BB/ W01355X/1 ), and the Louis-Jeantet Foundation . Work in the Higgins lab is funded by the CRUK RadNet Oxford and CRUK Clinician Scientist funding ( C34326/A19590 ). S.S.-B. was the recipient of an EMBO LT Fellowship ( ALTF 707-2019 ) and an MSCA-IF ( 886577 ). Publisher Copyright: © 2022 The Author(s)

PY - 2022/12/15

Y1 - 2022/12/15

N2 - POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

AB - POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

UR - http://www.scopus.com/inward/record.url?scp=85143857449&partnerID=8YFLogxK

U2 - 10.1016/j.molcel.2022.11.008

DO - 10.1016/j.molcel.2022.11.008

M3 - Article

C2 - 36455556

SN - 1097-2765

VL - 82

SP - 4664-4680.e9

JO - MOLECULAR CELL

JF - MOLECULAR CELL

IS - 24

ER -