Open AccessRejoining of DNA double‐strand breaks in vitro by single‐strand annealing
Author(s) -
Göttlich Bernd,
Reichenberger Susanne,
Feldmann Elke,
Pfeiffer Petra
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2580387.x
Subject(s) - dna ligase , ku80 , biology , ku70 , non homologous end joining , dna , exonuclease , microbiology and biotechnology , dna clamp , dna repair , d loop , dna polymerase , endonuclease , genetics , dna binding protein , gene , polymerase chain reaction , mitochondrial dna , transcription factor , reverse transcriptase
Nonhomologous DNA end joining (NHEJ) is considered the major pathway of double‐strand break (DSB) repair in vertebrate cells. Various studies indicated the existence of at least two different NHEJ pathways; one that joins DNA ends accurately and depends on Ku, a protein heterodimer that binds to DNA ends, and one that generates deletions and is independent of Ku. While the former pathway has been characterised in some detail, only little is known about the latter error‐prone. We have partially purified such an NHEJ activity from extracts of Xenopus laevis eggs. End‐joined junctions formed in the most extensively purified protein fraction displayed deletions containing short patches of sequence homology at their break points, a feature characteristic of single‐strand annealing (SSA). Detailed biochemical characterisation revealed the presence of DNA ligase III, DNA polymerase ε, FEN‐1 endonuclease, and exonuclease activities of 5′‐3′ and 3′‐5′ directionality. We show that these activities are able to correctly process proposed intermediates of SSA. Interestingly, neither Ku nor the associated DNA‐dependent protein kinase were detected, indicating that the mechanism can dispense with Ku. Our findings provide evidence for the existence of an error‐prone NHEJ pathway that creates deletions by microhomology‐driven SSA.