Promotion of double-strand break repair by human nuclear extracts preferentially involves recombination with intact homologous DNA

Abstract

Parameters of DNA double strand break (dsb) repair catalysed by human nuclear extract were analysed using, as substrate, the replicative form (RF) of M13 mp8 in which a single double strand break (dsb) was introduced by restriction. After incubation with the extract, the dsb repair was estimated by the ability of the incubated RF to produce plaques following transfection into JM 109 (Rec A-) bacteria. The possibility of recombination with a purified fragment from M13 mp8 RF enhances up to 20 times the plaquing ability of the RF. The repair by recombination occurs under several conditions: i) the break in the RF must be located in the region of homology with the fragment. ii) the fragment has to be intact in the region corresponding to the break in the RF. iii) a minimal length of homology between the region surrounding the dsb in the RF, and the fragment is required. The in vitro reaction is ATP dependent and dNTP's partially dependent. Dephosphorylation of the free ends in the RF decreases the repair by ligation but is without effect on the recombination.

Expression of ATM in ataxia telangiectasia fibroblasts rescues defects in DNA double‐strand break repair in nuclear extracts

“…Extract preparation was based on that of Lopez et al [1987] with some modification. Ice-cold conditions were used throughout.…”

Human POMp75 is identified as the pro-oncoprotein TLS/FUS: both POMp75 and POMp100 DNA homologous pairing activities are associated to cell proliferation

“…Nuclei from 2 to 5610 8 cells were isolated by hypotonic lysis and nuclear extracts were prepared as described previously (Lopez and Coppey, 1987).…”

A system for assaying homologous recombination at the endogenous human thymidine kinase gene.

“…Of particular interest is the reduction to homozygosity of mutations at tumorsuppressor loci as the cause of a proportion of retinoblastomas, osteosarcomas (1)(2)(3), Wilm tumors (4)(5)(6), astrocytomas (7), and meningiomas and acoustic neuromas (8,9). Previous analyses of recombination in cultured mammalian cells have used extrachromosomal plasmid-based systems or integrated markers in tandem array (10)(11)(12)(13)(14)(15)(16)(17), but these may not be appropriate models for recombinational events between autosomal genes in their native chromosomal environment. The identification of interchromosomal homologous recombination in mammalian cells in vitro has been largely confined to detecting post-S-phase recombination and the reduction to homozygosity of large chromosomal regions where the result is loss of a dominant (usually wild-type) allele or the identification ofrecombination-induced cytogenetic changes (18)(19)(20)(21).…”