PNNL

Abstract

Limited nucleotide excision repair (NER) requires at least ~40 proteins in extracts from purified proteins (1,2), although perhaps hundreds of proteins may influence DNA repari in cells. For efficient DNA repair in extracts, it is important to utilize a system containing large quantities of active DNA repair proteins uncontaminated with nonsepcific nucleases. Unlike extracts from mammalian cells that repari ~2% of the input DNA, both injected Xenopus oocytes (3) and oocyte nuclear extracts can repair ~100% of the input damaged DNA by NER with little or no synthesis on undamaged control substrate. Repair activity in extracts can be inactivated with antibodies and/or inhibitors, and then repair can be restored by addition of exoggenous proteins (4). A further advantage of the Xenopus system is that results obtained from injection experiments in living cells can be compared to results obtained in nuclear extracts.