An intramolecular FRET system monitors fingers subdomain opening in Klentaq1

@article{citeulike:2414119, abstract = {A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. However, this FRET system was limited in that it could not directly measure the rate of fingers subdomain opening by FRET after polymerization or in the absence of DNA. Here we report the development of a new system capable of measuring both fingers subdomain closure and reopening by FRET, and show that the rate of fingers subdomain opening is limited only by the rate of polymerization. We anticipate that this system will scale down to the single molecule level, allowing measurement of fingers subdomain movements in the presence of incorrect nucleotide and in the absence of DNA. 10.1110/ps.073309208}, author = {Allen, William J. and Rothwell, Paul J. and Waksman, Gabriel }, citeulike-article-id = {2414119}, doi = {http://dx.doi.org/10.1110/ps.073309208}, journal = {Protein Sci}, keywords = {domain, fret, invivo, subunit}, month = {March}, number = {3}, pages = {401--408}, posted-at = {2008-02-22 12:42:25}, priority = {3}, title = {An intramolecular FRET system monitors fingers subdomain opening in Klentaq1}, url = {http://dx.doi.org/10.1110/ps.073309208}, volume = {17}, year = {2008} }

TY - JOUR ID - citeulike:2414119 L3 - citeulike-article-id:2414119 TI - An intramolecular FRET system monitors fingers subdomain opening in Klentaq1 JF - Protein Sci VL - 17 IS - 3 SP - 401 EP - 408 N2 - A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. However, this FRET system was limited in that it could not directly measure the rate of fingers subdomain opening by FRET after polymerization or in the absence of DNA. Here we report the development of a new system capable of measuring both fingers subdomain closure and reopening by FRET, and show that the rate of fingers subdomain opening is limited only by the rate of polymerization. We anticipate that this system will scale down to the single molecule level, allowing measurement of fingers subdomain movements in the presence of incorrect nucleotide and in the absence of DNA. 10.1110/ps.073309208 KW - domain KW - fret KW - invivo KW - subunit AU - Allen, William AU - Rothwell, Paul AU - Waksman, Gabriel PY - 2008/03/01/ UR - http://dx.doi.org/10.1110/ps.073309208 ER -