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Papers In Press, published online ahead of print July 24, 2003
Department of Biochemistry, Physics and Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-8725
Corresponding Author: arun.alphonse{at}vanderbilt.edu
The initial high affinity binding of ssDNA by replication protein A (RPA) involves the tandem domains in the central region of the RPA70 subunit (RPA70AB). However, it was not clear if the two domains, RPA70A and RPA70B, bind DNA simultaneously or sequentially. Here, using primarily heteronuclear NMR complemented by fluorescence spectroscopy, we have analyzed the binding characteristics of the individual RPA70A and RPA70B domains and compared them to the intact RPA70AB. NMR chemical shift comparisons confirmed that RPA70A and RPA70B tumble independently in solution in the absence of ssDNA. NMR chemical shift perturbations showed that all ssDNA oligomers bind to the same sites as observed in the X-ray crystal structure of RPA70AB complexed to d(C)8. Titrations using a variety of 5’mer ssDNA oligomers showed that RPA70A has a 5-10 fold higher affinity for ssDNA than RPA70B. Detailed analysis of ssDNA binding to RPA70A revealed that all DNA sequences interact in a similar mode. Fluorescence binding measurements with a variety of 8-10’mer DNA sequences showed that RPA70AB interacts with DNA with approximately 100-fold higher affinity than the isolated domains. Calculation of the theoretical “linkage effect” from the structure of RPA70AB suggests the high overall affinity for ssDNA is a by-product of the covalent attachment of the two domains via a short flexible tether, which increases the effective local concentration. Taken together, our data are consistent with a sequential model of DNA binding by RPA according to which RPA70A binds the majority of DNA first and subsequent loading of RPA70B domain is facilitated by the linkage effect.
J. Biol. Chem, 10.1074/jbc.M305871200
Submitted on June 4, 2003
Revised on July 24, 2003
Accepted on July 24, 2003
Independent and coordinated functions of the replication protein A tandem high affinity ssDNA binding domains
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