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Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates
Author(s) -
Gyimesi Máté,
Pires Ricardo H.,
Billington Neil,
Sarlós Kata,
Kocsis Zsuzsa S.,
Módos Károly,
Kellermayer Miklós S. Z.,
Kovács Mihály
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.13-234088
Subject(s) - homologous recombination , helicase , dna , chemistry , monomer , bloom syndrome , dna repair , biophysics , holliday junction , biology , gene , biochemistry , rna , organic chemistry , polymer
Homologous recombination (HR) is a key process in the repair of double‐stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Bloom's syndrome RecQ‐family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR‐intermediate DNA structures, by using single‐molecule visualization (electron microscopic and atomic force microscopic single‐particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full‐length BLM, we used a previously characterized truncated construct (BLM 642–1290 ) as a monomeric control. Contrary to previous models proposing a ring‐forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single‐stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.—Gyimesi, M., Pires, R.H., Billington, N., Sarlós, K., Kocsis, Z.S., Módos, K., Kellermayer, M. S. Z., Kovács, M., Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates. FASEB J. 27, 4954–4964 (2013). www.fasebj.org