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Mechanistic study on binding of human Rad54 to nucleosome
Author(s) -
Wang Yuzhen,
Tsai YuCheng,
UrenaRivera Esteban,
Chen Junghuei
Publication year - 2008
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/fasebj.22.1_supplement.989.5
Subject(s) - nucleosome , naked dna , dna , biophysics , chemistry , binding site , microbiology and biotechnology , biology , chromatin , biochemistry , plasmid
How to make DNA accessible to hRad51 or hRad51‐ssDNA filament during HR process is still a puzzle, although several studies showed that Rad54 exhibit chromatin remodeling activity. Here we explore interaction of Rad54 with nucleosomes. N‐His‐tagged human Rad54 and N‐His plus C‐Flag‐tagged human Rad54B were purified through baculovirus system. Various biochemical assays were performed to characterize hRad54 and hRad54B. We found that hRad54's ATPase activitity is more efficient than hRad54B's, and significantly decreased in the presence of nucleosome, no such change was observed with Rad54B. We also found that hRad54, not hRad54B, exhibits strong binding activity to nucleosomes, preferentially binding to high order aggregates of nucleosomes in low concentration. No such strong binding shift on naked DNA was observed with either hRad54 or hRad54B in the same condition. We are currently preparing N‐terminal truncated Rad54 which lack the primary binding site of Rad51, and C‐terminal truncated Rad54 with deletion of positively charged surface patch, and several single mutants selected in conserved DNA binding site and bi‐lobe ATPase core to further characterize the binding mechanism of hRad54 with nucleosomes. In addition, the discrepancy in ATPase activity and nucleosome binding between hRad54 and Rad54B is being confirmed with untagged proteins.