Nonhomologous DNA end joining of synthetic hairpin substrates inXenopus laevisegg extracts

Abstract

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'-or 3'-protruding single-strands (PSS) of arbitrary sequence and length.

A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA

“…We show that the activity cleaves off oligonucleotides of S Reichenberger et al 356 Genes to Cells, (1996) Fig. 4A and Beyert et al 1994). The asterisks mark the 5 0 -hydroxyl groups that are [ 32 P]-phosphorylated during substrate preparation.…”

“…Recently, we have developed entirely synthetic hairpin-shaped oligonucleotides as model substrates that facilitate a more systematic analysis of end joining reactions because they allow the design of freely variable DSB termini (Beyert et al 1994). The general concept of hairpin construction from two oligonucleotide building blocks is outlined in Fig.…”

“…During ligation of the stem oligo to the linker oligo, a [ 32 P]-phosphate label is incorporated into the duplex stem. Since end joining in Xenopus laevis egg extracts requires a minimal duplex stem length of at least 2.5 double-helical turns (Beyert et al 1994) the duplex portions of all hairpins used in this study have about 3.0 and 3.5 double-helical turns. End joining is monitored as an intermolecular reaction between two hairpin molecules at their open ends to generate dimeric dumbellshaped products.…”

“…The protocol for hairpin construction from a constant stem oligo and a variable linker oligo as well as the sequences of these oligos have been described in detail previously (Beyert et al 1994). In brief: all oligonucleotides used for hairpin construction were purified over preparative sequencing gels to remove smaller chain termination products.…”

“…The capacities of this particular end joining system have been studied with specialized substrate types (synthetic hairpin-shaped oligonucleotides) that permit the construction of DSB-termini of freely variable sequence and structure. Use of these hairpin substrates showed that successful end joining in the Xenopus in vitro system requires a minimal DNA duplex length of 27 bp and ceases when the length of the overhangs exceeds 10-11 nt (Beyert et al 1994).…”