Journal Article

BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair

Joanna R. Morris and Ellen Solomon

in Human Molecular Genetics

Volume 13, issue 8, pages 807-817
Published in print April 2004 | ISSN: 0964-6906
Published online February 2004 | e-ISSN: 1460-2083 | DOI: http://dx.doi.org/10.1093/hmg/ddh095
BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair

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The N-terminus of the BRCA1 protein bears a RING finger domain that functions as an E3 ubiquitin ligase in vitro where it is able to catalyse the synthesis of monoubiquitin and polyubiquitin targeted proteins. This activity is greatly increased when BRCA1 is in a complex with its N-terminal binding partner BARD1. In this report we use an immunohistochemical approach to demonstrate the association of cellular BRCA1 with the end product of the ubiquitin conjugation and ligation pathway, conjugated ubiquitin. Association is apparent at DNA replication structures in S-phase and following treatment with hydroxyurea and also at sites of double strand break repair after exposure to ionizing radiation. Down-regulation of endogenous, cellular BRCA1 : BARD1 using siRNA results in abrogation of ubiquitin conjugation in these structures, suggesting that heterodimer activity is required for their formation. Conversely, ectopically expressed full-length BRCA1, but not BRCA1 bearing specific N-terminal amino acid substitutions, is able to cooperate with BARD1 to increase ubiquitin conjugation in cells. Conjugation of ubiquitin in foci is inhibited by the expression of ubiquitin bearing a lysine 6 mutation suggesting that the ubiquitin polymers formed at these sites are dependent on lysine-6 for linkage. Together these data demonstrate that BRCA1 directed ligation of ubiquitin occurs during S-phase and in response to replication stress and DNA damage and is therefore likely to be a significant aspect of BRCA1 cellular activity.

Journal Article.  7167 words.  Illustrated.

Subjects: Genetics and Genomics

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