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Published online 19 April 2004

Nucleic Acids Research, 2004, Vol. 32, No. 7 2158-2170
© 2004 Oxford University Press

Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A

Sheng Cui, Daniele Arosio, Kevin M. Doherty1, Robert M. Brosh Jr1, Arturo Falaschi and Alessandro Vindigni*

International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy and 1 Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA

*To whom correspondence should be addressed. Tel: +39 040 3757326; Fax: +39 040 226555; Email: vindigni{at}icgeb.org

Received December 23, 2003; Revised and Accepted March 23, 2004

RecQ helicases are required for the maintenance of genome stability. Characterization of the substrate specificity and identification of the binding partners of the five human RecQ helicases are essential for understanding their function. In the present study, we have developed an efficient baculovirus expression system that allows us to obtain milligram quantities of recombinant RECQ1. Our gel filtration and dynamic light scattering experiments show that RECQ1 has an apparent molecular mass of 158 kDa and a hydrodynamic radius of 5.4 ± 0.6 nm, suggesting that RECQ1 forms dimers in solution. The oligomeric state of RECQ1 remains unchanged upon binding to a single-stranded (ss)DNA fragment of 50 nt. We show that RECQ1 alone is able to unwind short DNA duplexes (<110 bp), whereas considerably longer substrates (501 bp) can be unwound only in the presence of human replication protein A (hRPA). The same experiments with Escherichia coli SSB show that RECQ1 is specifically stimulated by hRPA. However, hRPA does not affect the ssDNA-dependent ATPase activity of RECQ1. In addition, our far western, ELISA and co-immunoprecipitation experiments demonstrate that RECQ1 physically interacts with the 70 kDa subunit of hRPA and that this interaction is not mediated by DNA.


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