Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • R Jessberger - , Stanford Medicine (Author)
  • Paul Berg - , Stanford Medicine (Author)

Abstract

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.

Details

Original languageEnglish
Pages (from-to)445-57
Number of pages13
JournalMolecular and cellular biology
Volume11
Issue number1
Publication statusPublished - Jan 1991
Peer-reviewedYes
Externally publishedYes

External IDs

PubMedCentral PMC359648
Scopus 0025968004

Keywords

Keywords

  • Animals, Cell Line, Cell Nucleus/physiology, Chromosome Deletion, DNA Damage, DNA Repair, Humans, Polymerase Chain Reaction, Recombination, Genetic, Transfection