Received for publication November 28, 2006.
Revised January 4, 2007.
Accepted for publication January 5, 2007.

Differential selection of acridine resistance mutations in human DNA topoisomerase II is dependent on the acridine structure

Abstract

Type II DNA topoisomerases are targets of acridine drugs. Nine mutations conferring resistance to acridines were obtained by forced molecular evolution, using mAMSA, mAMCA, AMCA and DACA as selection agents. Mutations H514Y, E522K, G550R, A596T, Y606C, R651C and D661N were in the B' domain, G465D and P732L were not. With AMCA four mutations were selected (E522K, G550R, A596T and D661N). Two mutations were selected with mAMCA (Y606C and R651C) and two with mAMSA (G465D and P732L). Interestingly there was no overlap between mutation selection with AMCA and mAMSA or mAMCA. AMCA lacks the methoxy substituent present in mAMCA and mAMSA, suggesting this motif determines the mutations selected. With the fourth acridine DACA, five mutations were selected for resistance (G465D, H514Y, G550R, A596T and D661N). G465D was selected with both DACA and mAMSA, and G550R, A596T and D661N were selected with both DACA and AMCA. DACA lacks the anilino motif of the other three drugs but retains the acridine ring motif. The overlap in selection with DACA and mAMSA or AMCA suggests that altered recognition of the acridine moiety may be involved in these mutations. We used Restriction Fragment Length Polymorphisms and heteroduplex analysis to demonstrate some mutations were selected multiple times (G465D, E522K, G550R, A596T and D661N), whilst others were selected only once (H514Y, Y606C, R651C and P732L). Here we compare the drug resistance profile of all nine mutations and report the biochemical characterisation of three, G550R, Y606C and D661N.

Key words: Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches, Topoisomerases