Polθ promotes the repair of 5′-DNA-protein crosslinks by microhomology-mediated end-joining

Gurushankar Chandramouly; Shuren Liao; Timur Rusanov; Nikita Borisonnik; Marissa L. Calbert; Tatiana Kent; Katherine Sullivan-Reed; Umeshkumar Vekariya; Ekaterina Kashkina; Tomasz Skorski; Hong Yan; Richard T. Pomerantz

doi:10.1016/j.celrep.2021.108820

Polθ promotes the repair of 5′-DNA-protein crosslinks by microhomology-mediated end-joining

Gurushankar Chandramouly, Shuren Liao, Timur Rusanov, Nikita Borisonnik, Marissa L. Calbert, Tatiana Kent, Katherine Sullivan-Reed, Umeshkumar Vekariya, Ekaterina Kashkina, Tomasz Skorski, Hong Yan, Richard T. Pomerantz

Research output: Contribution to journal › Article › peer-review

17 Scopus citations

Abstract

DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.

Original language	English
Article number	108820
Pages (from-to)	108820
Journal	Cell Reports
Volume	34
Issue number	10
DOIs	https://doi.org/10.1016/j.celrep.2021.108820
State	Published - Mar 9 2021

Keywords

Cell Line
Xenopus/growth & development
DNA Polymerase theta
Humans
Formaldehyde/pharmacology
Phosphoric Diester Hydrolases/genetics
Animals
DNA/chemistry
DNA-Binding Proteins/genetics
RNA, Guide, CRISPR-Cas Systems
Mice
Cross-Linking Reagents/pharmacology
DNA End-Joining Repair/drug effects
DNA-Directed DNA Polymerase/deficiency
Embryonic Stem Cells/cytology
Ovum/metabolism

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10.1016/j.celrep.2021.108820

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title = "Polθ promotes the repair of 5′-DNA-protein crosslinks by microhomology-mediated end-joining",

abstract = "DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.",

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author = "Gurushankar Chandramouly and Shuren Liao and Timur Rusanov and Nikita Borisonnik and Calbert, {Marissa L.} and Tatiana Kent and Katherine Sullivan-Reed and Umeshkumar Vekariya and Ekaterina Kashkina and Tomasz Skorski and Hong Yan and Pomerantz, {Richard T.}",

year = "2021",

month = mar,

day = "9",

doi = "10.1016/j.celrep.2021.108820",

language = "English",

volume = "34",

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journal = "Cell Reports",

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T1 - Polθ promotes the repair of 5′-DNA-protein crosslinks by microhomology-mediated end-joining

AU - Chandramouly, Gurushankar

AU - Liao, Shuren

AU - Rusanov, Timur

AU - Borisonnik, Nikita

AU - Calbert, Marissa L.

AU - Kent, Tatiana

AU - Sullivan-Reed, Katherine

AU - Vekariya, Umeshkumar

AU - Kashkina, Ekaterina

AU - Skorski, Tomasz

AU - Yan, Hong

AU - Pomerantz, Richard T.

PY - 2021/3/9

Y1 - 2021/3/9

N2 - DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.

AB - DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.

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KW - Xenopus/growth & development

KW - DNA Polymerase theta

KW - Humans

KW - Formaldehyde/pharmacology

KW - Phosphoric Diester Hydrolases/genetics

KW - Animals

KW - DNA/chemistry

KW - DNA-Binding Proteins/genetics

KW - RNA, Guide, CRISPR-Cas Systems

KW - Mice

KW - Cross-Linking Reagents/pharmacology

KW - DNA End-Joining Repair/drug effects

KW - DNA-Directed DNA Polymerase/deficiency

KW - Embryonic Stem Cells/cytology

KW - Ovum/metabolism

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DO - 10.1016/j.celrep.2021.108820

M3 - Article

C2 - 33691100

SN - 2211-1247

VL - 34

SP - 108820

JO - Cell Reports

JF - Cell Reports

IS - 10

M1 - 108820

ER -

Polθ promotes the repair of 5′-DNA-protein crosslinks by microhomology-mediated end-joining

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