Resolution of Holliday junction substrates by human topoisomerase I.

@article{citeulike:2937803, abstract = {Prompted by the close relationship between tyrosine recombinases and type IB topoisomerases we have investigated the ability of human topoisomerase I to resolve the typical intermediate of recombinase catalysis, the Holliday junction. We demonstrate that human topoisomerase I catalyzes unidirectional resolution of a synthetic Holliday junction substrate containing two preferred cleavage sites surrounded by DNA sequences supporting branch migration. Deleting part of the N-terminal domain (amino acid residues 1-202) did not affect topoisomerase I resolution activity, whereas a topoisomerase I variant lacking both the N-terminal domain and amino acid residues 660-688 of the linker domain was unable to resolve the Holliday junction substrate. The inability of the double deleted variant to mediate resolution correlated with the inability of this enzyme to introduce concomitant cleavage at the two preferred cleavage sites in a single Holliday junction substrate, which is a prerequisite for resolution. As determined by the gel electrophoretic mobility of native enzyme or enzyme crosslinked by disulfide bridging, the double deleted mutant existed almost entirely in a dimeric form. The impairment of this enzyme in performing double cleavages on the Holliday junction substrate may be explained by only one cleavage competent active site being formed at a time within the dimer. The assembly of only one active site within dimers is a well-known characteristic of the tyrosine recombinases. Hence, the obtained results may suggest a recombinase-like active site assembly of the double deleted topoisomerase I variant. Taken together the presented results consolidate the relationship between type IB topoisomerases and tyrosine recombinases.}, address = {Department of Molecular Biology, University of Aarhus, C.F. Møllers All\'{e}, Building 130, DK-8000, Aarhus C, Denmark.}, author = {Hede, M. S. and Petersen, R. L. and Frøhlich, R. F. and Kr\"{u}ger, D. and Andersen, F. F. and Andersen, A. H. and Knudsen, B. R. }, citeulike-article-id = {2937803}, doi = {http://dx.doi.org/10.1016/j.jmb.2006.10.050}, issn = {0022-2836}, journal = {Journal of molecular biology}, keywords = {knudsen, topoisomerasi}, month = {January}, number = {4}, pages = {1076--1092}, posted-at = {2008-06-27 18:23:01}, priority = {2}, title = {Resolution of Holliday junction substrates by human topoisomerase I.}, url = {http://dx.doi.org/10.1016/j.jmb.2006.10.050}, volume = {365}, year = {2007} }

TY - JOUR ID - citeulike:2937803 L3 - citeulike-article-id:2937803 TI - Resolution of Holliday junction substrates by human topoisomerase I. JF - Journal of molecular biology VL - 365 IS - 4 SP - 1076 EP - 1092 AD - Department of Molecular Biology, University of Aarhus, C.F. Møllers Allé, Building 130, DK-8000, Aarhus C, Denmark. SN - 0022-2836 N2 - Prompted by the close relationship between tyrosine recombinases and type IB topoisomerases we have investigated the ability of human topoisomerase I to resolve the typical intermediate of recombinase catalysis, the Holliday junction. We demonstrate that human topoisomerase I catalyzes unidirectional resolution of a synthetic Holliday junction substrate containing two preferred cleavage sites surrounded by DNA sequences supporting branch migration. Deleting part of the N-terminal domain (amino acid residues 1-202) did not affect topoisomerase I resolution activity, whereas a topoisomerase I variant lacking both the N-terminal domain and amino acid residues 660-688 of the linker domain was unable to resolve the Holliday junction substrate. The inability of the double deleted variant to mediate resolution correlated with the inability of this enzyme to introduce concomitant cleavage at the two preferred cleavage sites in a single Holliday junction substrate, which is a prerequisite for resolution. As determined by the gel electrophoretic mobility of native enzyme or enzyme crosslinked by disulfide bridging, the double deleted mutant existed almost entirely in a dimeric form. The impairment of this enzyme in performing double cleavages on the Holliday junction substrate may be explained by only one cleavage competent active site being formed at a time within the dimer. The assembly of only one active site within dimers is a well-known characteristic of the tyrosine recombinases. Hence, the obtained results may suggest a recombinase-like active site assembly of the double deleted topoisomerase I variant. Taken together the presented results consolidate the relationship between type IB topoisomerases and tyrosine recombinases. KW - knudsen KW - topoisomerasi AU - Hede, MS AU - Petersen, RL AU - Frøhlich, RF AU - Krüger, D AU - Andersen, FF AU - Andersen, AH AU - Knudsen, BR PY - 2007/01/26/ UR - http://dx.doi.org/10.1016/j.jmb.2006.10.050 ER -