MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA

Ka Cheong Lee; Kay Padget; Hannah Curtis; Ian Cowell; Davide Moiani; Zbyslaw Sondka; Nicholas Morris; Graham Jackson; Simon Cockell; John Tainer; Caroline Austin

doi:10.1242/bio.20121834

MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA

Ka Cheong Lee, Kay Padget, Hannah Curtis, Ian Cowell, Davide Moiani, Zbyslaw Sondka, Nicholas Morris, Graham Jackson, Simon Cockell, John Tainer, Caroline Austin^*

^*Corresponding author for this work

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

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Abstract

Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIalpha from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIalpha and beta complex levels formed in the absence of an anti-topoisomerase II drug

Original language	English
Pages (from-to)	863-873
Journal	Biology Open
Volume	1
Issue number	9
Early online date	11 Jul 2012
DOIs	https://doi.org/10.1242/bio.20121834
Publication status	Published - 15 Sept 2012

Keywords

Topoisomerase II
MRE11
DSB repair
Protein-DNA adducts
A-TLD

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Padget K Output 3 MRE11Final published version, 699 KBLicence: CC BY-NC-SA

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title = "MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA",

abstract = "Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIalpha from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIalpha and beta complex levels formed in the absence of an anti-topoisomerase II drug",

keywords = "Topoisomerase II, MRE11, DSB repair, Protein-DNA adducts, A-TLD",

author = "Lee, \{Ka Cheong\} and Kay Padget and Hannah Curtis and Ian Cowell and Davide Moiani and Zbyslaw Sondka and Nicholas Morris and Graham Jackson and Simon Cockell and John Tainer and Caroline Austin",

note = "Funding information: This work was funded by LRF grant 04037 and LLR grant 07038 (K.C.L., K.P., H.C., I.G.C., Z.S., G.H.J., N.J.M., S.J.C. and C.A.A.). The efforts of D.M. and J.A.T. on Mre11 mutant preparations were supported by National Cancer Institute grant CA117638 (J.A.T.).",

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AU - Lee, Ka Cheong

AU - Padget, Kay

AU - Curtis, Hannah

AU - Cowell, Ian

AU - Moiani, Davide

AU - Sondka, Zbyslaw

AU - Morris, Nicholas

AU - Jackson, Graham

AU - Cockell, Simon

AU - Tainer, John

AU - Austin, Caroline

N1 - Funding information: This work was funded by LRF grant 04037 and LLR grant 07038 (K.C.L., K.P., H.C., I.G.C., Z.S., G.H.J., N.J.M., S.J.C. and C.A.A.). The efforts of D.M. and J.A.T. on Mre11 mutant preparations were supported by National Cancer Institute grant CA117638 (J.A.T.).

PY - 2012/9/15

Y1 - 2012/9/15

N2 - Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIalpha from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIalpha and beta complex levels formed in the absence of an anti-topoisomerase II drug

AB - Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIalpha from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIalpha and beta complex levels formed in the absence of an anti-topoisomerase II drug

KW - Topoisomerase II

KW - MRE11

KW - DSB repair

KW - Protein-DNA adducts

KW - A-TLD

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U2 - 10.1242/bio.20121834

DO - 10.1242/bio.20121834

M3 - Article

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SP - 863

EP - 873

JO - Biology Open

JF - Biology Open

IS - 9

ER -

MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA

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