JBC 2006 Abstract

DNA-dependent activation of ATM kinase in chromatin-free nuclear extracts

Yuan-Chin Tsai and Leroy F. Liu*

Department of Pharmacology

UMDNJ-Robert Wood Johnson Medical School

675 Hoes Lane

Piscataway, NJ 08854

USA

Summary

A chromatin-free nuclear extract capable of supporting ATM activation in response to DNA double-strand breaks (DSBs) was developed. Addition of short duplex DNA fragments (e.g. 30-mer) incapable of forming nucleosomes was shown to efficiently activate ATM kinase in the nuclear extract, as evidenced by the stimulation of ATM autophosphorylation at Ser-1981, and phosphorylation of ATM substrates such as 53BP1, Chk1 (Ser-317), Chk2 (Ser-33/35) and p53 (Ser-15). Single-stranded oligonucleotides (ODNs) as short as 30-mer also efficiently activated ATM kinase in the in vitro system. Moreover, DNA substrates containing single-stranded regions such as gaps and flaps also effectively activated ATM kinase. These in vitro results suggest that the chromatin structure is not obligatory for ATM activation, and altered DNA structures other than double-strand breaks can also efficiently activate ATM kinase.

posted by yuanchin @ 8:37 PM 0 comments