Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage

Bibliographic Details
Main Author:	Russell, Beatriz
Language:	English
Published:	University of Cincinnati / OhioLINK 2009
Subjects:	Molecular Biology BLM II¿¿¿¿ topoisomerase II¿¿¿¿ topoisomerase BLM and topoisomerase II¿¿¿¿ BLM and topoisomerase helicase
Online Access:	http://rave.ohiolink.edu/etdc/view?acc_num=ucin1250676966

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spelling	ndltd-OhioLink-oai-etd.ohiolink.edu-ucin12506769662021-08-03T06:13:37Z Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage Russell, Beatriz Molecular Biology BLM II¿¿¿¿ topoisomerase II¿¿¿¿ topoisomerase BLM and topoisomerase II¿¿¿¿ BLM and topoisomerase helicase The BLM helicase is a member of the RecQ-like family of 3’-5’ ATP- and Mg2+-dependent helicases. Cells deficient in BLM are characterized by increased sister chromatid exchanges, quadriradial structures and chromosome breaks, chromosome structures that suggest the disruption of normal mechanisms that resolve recombination intermediates and maintain chromosome stability. Previously identified interactions between yeast RecQ-like helicases and topoisomerases suggest cooperation in DNA transactions such as recombination repair and chromosome segregation. This work demonstrates that human BLM and topoisomerase IIα interact directly. BLM and topoisomerase IIα co-immunoprecipitate from human cells and co-localize in a cell cycle-specific manner. Their association and co-localization increases in S- and G2/M- and is predominant in M-phase. In vitro binding assays demonstrate that amino acids 489-587 within the N-terminus of BLM are required for this interaction. In vitro biochemical analysis of the decatenation activity of topoisomerase IIα in the presence of BLM revealed no effect by BLM. On the other hand, BLM helicase activity is enhanced approximately three- and five-fold by topoisomerase IIα on a 3’ overhang duplex and bubble substrates, respectively, but not an X-junction substrate. These data suggest that BLM and topoisomerase IIα may be involved in the processing of early homologous replication intermediates, but not structures that form later. In vivo, siRNA mediated knock-down of either BLM, topoisomerase IIα, or both in combination increased chromosome breakage to the same extent compared to a scrambled control knock-down, suggesting a common pathway that requires BLM and topoisomerase IIα for protecting the integrity of genomic DNA. In order to determine the importance of the BLM-topoisomerase IIα interaction, a mutant BLM protein lacking the topoisomerase IIα interaction domain (aa 489-587) was generated, EGFP-BLMΔ489-587. The mutant protein was unable to co-immunoprecipitate topoisomerase IIα, although it still localized to nuclear PML foci and the nucleolus as wild-type BLM. To determine the significance of their interaction on chromosome breakage, the ability of this mutant to reduce the high endogenous levels of γH2AX foci of BS cells was tested. Transfection BS cells line with the EGFP-BLMΔ489-587 mutant protein did not reduce γH2AX foci formation to the levels of transfection with a wild-type BLM protein, but exacerbated the number of γH2AX foci in cells compared to untransfected cells. We propose that in the presence of BLM, cells commit to a BLM dependent pathway of double strand break repair and the inability of EGFP-BLMΔ489-587 mutant to bind topoisomerase IIα locks the cells in such pathways without being able to correctly finish the repair. Taken together, these data suggest an involvement of BLM and topoisomerase IIα in a pathway that prevents chromosome breakage. The in vitro biochemical data suggest that this pathway may involve the regulation of homologous recombination through the processing of DNA intermediates occurring at early stages of repair. 2009 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin1250676966 http://rave.ohiolink.edu/etdc/view?acc_num=ucin1250676966 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
collection	NDLTD
language	English
sources	NDLTD
topic	Molecular Biology BLM II¿¿¿¿ topoisomerase II¿¿¿¿ topoisomerase BLM and topoisomerase II¿¿¿¿ BLM and topoisomerase helicase
spellingShingle	Molecular Biology BLM II¿¿¿¿ topoisomerase II¿¿¿¿ topoisomerase BLM and topoisomerase II¿¿¿¿ BLM and topoisomerase helicase Russell, Beatriz Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage
author	Russell, Beatriz
author_facet	Russell, Beatriz
author_sort	Russell, Beatriz
title	Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage
title_short	Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage
title_full	Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage
title_fullStr	Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage
title_full_unstemmed	Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage
title_sort	interactions with topoisomerase iia enhance the unwinding activity of the blm helicase on recombination substrates and are necessary for preventing chromosome breakage
publisher	University of Cincinnati / OhioLINK
publishDate	2009
url	http://rave.ohiolink.edu/etdc/view?acc_num=ucin1250676966
work_keys_str_mv	AT russellbeatriz interactionswithtopoisomeraseiiaenhancetheunwindingactivityoftheblmhelicaseonrecombinationsubstratesandarenecessaryforpreventingchromosomebreakage
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Interactions with topoisomerase IIa enhance the unwinding activity of the BLM helicase on recombination substrates and are necessary for preventing chromosome breakage

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