TY - JOUR
T1 - Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A
AU - Cui, Sheng
AU - Arosio, Daniele
AU - Doherty, Kevin M.
AU - Brosh, Robert M.
AU - Falaschi, Arturo
AU - Vindigni, Alessandro
N1 - Funding Information:
The assistance of Drs Raffaella Klima and Claudia Ortega in the expression of RECQ1 in insect cells is gratefully acknowledged. We are indebted to Dr Mark Wold for providing purified hRPA and the plasmid for the expression of hRPA in E.coli. The work was supported by a grant from the Human Frontier Science Program, by a FIRB grant of MIUR (Ministero dell'Istruzione dell'Universita' e della Ricerca) and by grant no. 02.00648.ST97 of the Consiglio Nazionale delle Ricerche, Rome.
PY - 2004
Y1 - 2004
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=2342487313&partnerID=8YFLogxK
U2 - 10.1093/nar/gkh540
DO - 10.1093/nar/gkh540
M3 - Article
C2 - 15096578
AN - SCOPUS:2342487313
SN - 0305-1048
VL - 32
SP - 2158
EP - 2170
JO - Nucleic acids research
JF - Nucleic acids research
IS - 7
ER -