Heat sensitivity of Hela S3 Cell DNA topoisomerase II

✍ Scribed by Raymond L. Warters; Louis R. Barrow

• Publisher: John Wiley and Sons
• Year: 1994
• Tongue: English
• Weight: 730 KB
• Volume: 159
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041590311

✦ Synopsis

Abstract

The sensitivity of HeLa DNA topoisomerase II to 45°C heat shock was measured both in the intact cell and in vitro. In the intact cell, DNA topoisomerase II activity was estimated by measuring the formation and reversal of enzyme‐DNA cleavable complexes by alkaline filter elution of cells exposed to the enzyme poison 4′‐(9‐acridinylamino)(methanesulfon‐m‐anisidide). In vitro enzymatic activity was estimated by measuring changes in the topological state of plasmid and kinetoplast DNA produced by sonicates of nuclei from previously heated cells. The capacity of the enzyme to form, or reverse, enzyme‐DNA cleavable complexes was inactivated during 45°C heating with a reciprocal slope of 120 or 15 min, respectively. In vitro estimates of the activity of the enzyme from previously heated cells indicated that the enzyme was inactivated with a reciprocal slope of 99, 45, and 21 min after 45, 46 and 47°C heating, respectively. DNA topoisomerase I activity was inactivated with a reciprocal slope of 130 min at 45°C. The cumulative results indicate that during 45°C heat shock, thermal inactivation of neither DNA topoisomerase I nor II is rate limiting for either cell survival or for DNA replication. While DNA topoisomerase II is resistant in situ to heat inactivation, in vivo assays indicate that the enzyme's capacity to function in the intact cell may be compromised by hyperthermic changes in the enzyme's environment. © 1994 wiley‐Liss, Inc.