1R49

Human topoisomerase I (Topo70) double mutant K532R/Y723F

Summary for 1R49

• Entry DOI: 10.2210/pdb1r49/pdb
• Related: 1A36 1EJ9 1K4S 1K4T 1LPQ 1TMC
• Descriptor: 5'-D(*AP*AP*AP*AP*AP*GP*AP*CP*TP*TP*AP*GP*AP*AP*AP*AP*AP*TP*TP*TP*TP*T)-3', 5'-D(P*AP*AP*AP*AP*AP*TP*TP*TP*TP*TP*CP*TP*AP*AP*GP*TP*CP*TP*TP*TP*TP*T)-3', DNA topoisomerase I, ... (4 entities in total)
• Functional Keywords: protein, dna, topoisomerase i, isomerase-dna complex, isomerase/dna
• Biological source: Homo sapiens (human)
• Cellular location: Nucleus, nucleolus: P11387
• Total number of polymer chains: 3
• Total formula weight: 83675.90

Authors

Interthal, H.,Quigley, P.M.,Hol, W.G.,Champoux, J.J. (deposition date: 2003-10-03, release date: 2003-12-16, Last modification date: 2024-02-14)

Primary citation

Interthal, H.,Quigley, P.M.,Hol, W.G.,Champoux, J.J.
The role of lysine 532 in the catalytic mechanism of human topoisomerase I.
J.Biol.Chem., 279:2984-2992, 2004

PubMed Abstract: Based on co-crystal structures of human topoisomerase I with bound DNA, Lys(532) makes a minor groove contact with the strongly preferred thymidine residue at the site of covalent attachment (-1 position). Replacement of Lys(532) with either arginine or alanine has essentially no effect on the sequence preference of the enzyme, indicating that this interaction is not required for the preference for a T at the -1 position. Although both the cleavage and religation activities of the K532R mutant enzyme are reduced, cleavage is reduced to a greater extent than religation. The reverse is true for the K532A mutant enzyme with religation so impaired that the nicked intermediate accumulates during plasmid relaxation assays. Consistent with the shift in the cleavage religation equilibrium toward cleavage for the K532A mutant enzyme, expression of the mutant enzyme in Saccharomyces cerevisiae is cytotoxic, and thus this mutant enzyme mimics the effects of the anticancer drug camptothecin. Cleavage assays with the mutant enzymes using an oligonucleotide containing a 5'-bridging phosphorothiolate indicate that Lys(532) functions as a general acid during cleavage to protonate the leaving 5'-oxygen. It is possible that the contact with the -1 base is important during catalysis to provide positional rigidity to the active site. The corresponding residues in the vaccinia virus topoisomerase and the tyrosine recombinases may have similar critical roles in catalysis.

PubMed: 14594810
DOI: 10.1074/jbc.M309959200

Experimental method

X-RAY DIFFRACTION (3.13 Å)