TY - JOUR
T1 - USP1-trapping lesions as a source of DNA replication stress and genomic instability
AU - Coleman, Kate E.
AU - Yin, Yandong
AU - Lui, Sarah Kit Leng
AU - Keegan, Sarah
AU - Fenyo, David
AU - Smith, Duncan J.
AU - Rothenberg, Eli
AU - Huang, Tony T.
N1 - Funding Information:
We thank members of the Huang and Rothenberg labs for technical assistance and critical discussions. We also thank the technical staff members of Applied Stem Cell Technologies for the derivation of HCT116 USP1 mutant cell lines used in this study and Genewiz for subcloning assistance. We would like to especially thank Malik Kahli and Peter Tonzi for technical assistance and guidance on preparation of Ok-seq libraries, Huijun Xue (E. Rothenberg Lab) for assistance in derivation of stable cell lines, and Ipsita Subudhi for initial characterization of cell lines. pBabe-puro-NLS-mCerulean-PCNA plasmid was a gift from Gergely Rona (M. Pagano Lab). K.E.C. was supported by an ACS Postdoctoral Fellowship (130453-PF-17-006-01-CCG). This work was supported by NIH grants: GM139610, ES025166, and ES031658 (T.T.H.); R35 GM134918 (D.S.); R35 GM134947, AI153040, and CA247773 (E.R.). This work was also supported by the V Foundation BRCA Research collaborative grant (E.R. and T.T.H.). Sequencing was performed at the NYU Genome Technology Center (RRID: SCR_017929). The Genome Technology Center and the Cytometry and Cell Sorting Laboratory shared resource is partially supported by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center.
Funding Information:
We thank members of the Huang and Rothenberg labs for technical assistance and critical discussions. We also thank the technical staff members of Applied Stem Cell Technologies for the derivation of HCT116 USP1 mutant cell lines used in this study and Genewiz for subcloning assistance. We would like to especially thank Malik Kahli and Peter Tonzi for technical assistance and guidance on preparation of Ok-seq libraries, Huijun Xue (E. Rothenberg Lab) for assistance in derivation of stable cell lines, and Ipsita Subudhi for initial characterization of cell lines. pBabe-puro-NLS-mCerulean-PCNA plasmid was a gift from Gergely Rona (M. Pagano Lab). K.E.C. was supported by an ACS Postdoctoral Fellowship (130453-PF-17-006-01-CCG). This work was supported by NIH grants: GM139610, ES025166, and ES031658 (T.T.H.); R35 GM134918 (D.S.); R35 GM134947, AI153040, and CA247773 (E.R.). This work was also supported by the V Foundation BRCA Research collaborative grant (E.R. and T.T.H.). Sequencing was performed at the NYU Genome Technology Center (RRID: SCR_017929). The Genome Technology Center and the Cytometry and Cell Sorting Laboratory shared resource is partially supported by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by “USP1-trapping”. We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.
AB - The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by “USP1-trapping”. We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.
UR - http://www.scopus.com/inward/record.url?scp=85127480268&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85127480268&partnerID=8YFLogxK
U2 - 10.1038/s41467-022-29369-3
DO - 10.1038/s41467-022-29369-3
M3 - Article
C2 - 35365626
AN - SCOPUS:85127480268
SN - 2041-1723
VL - 13
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 1740
ER -