The purpose of this post is to list somewhat similar
articles by Kmiec (NPRO) and M Dutreix (CNRS-Institut Curie, Section Recherche, Orsay, France).
It is assumed that the reader has read most of Kmiec's
abstracts.
M Dutreix - background abstract from 2002 - which
proves the point the ww scientists are still pursuing
the gene targeting paridigm.
A new method (GOREC) for directed mutagenesis and gene repair by homologous recombination
R Maurisse1,2, J-P Feugeas2, E Biet3, I Kuzniak2, P Leboulch2, M Dutreix3 and J-S Sun1
1Laboratoire de Biophysique, UMR8646 CNRS-Muséum National d'Histoire Naturelle, U201 INSERM, Paris, France
2EPI0111 INSERM, Institut Universitaire d'Hématologie, Hôpital Saint Louis, Paris, France
3UMR2027 CNRS-Institut Curie, Section Recherche, Orsay, France
Correspondence to: J-S Sun, Laboratoire de Biophysique, UMR8646 CNRS-Muséum National d'Histoire Naturelle, U201 INSERM, 43 rue Cuvier, 75231 Paris cedex 05, France
Abstract
Directed mutagenesis in mammalian cells has been the focus of intense research because of its promising application for gene correction and engineering. Both natural and modified oligonucleotides (ODN), RNA-DNA chimeric oligonucleotide (RDO) and small fragment DNA (SFHR), as well as vector DNA were used for promoting homologous replacement with varying success. It was recently shown that a triple helix-forming oligonucleotide (TFO) tethered to an oligonucleotide (donor DNA) can enhance mutagenesis by homologous recombination in cells. The basic idea is to accelerate homology search by oligonucleotide-directed triple helix formation in the vicinity of the target site for donor DNA. Here we describe a new method named GOREC (guided homologous recombination) which shares similar gene targeting, but has notable difference in the concept with the previous method. It is made of a homing device (TFO) and a donor DNA for effecting distinct functions. They are linked together by non-covalent or covalent interaction. This modular concept allows guidance of either an oligonucleotide (ODN, RDO) or a small DNA fragment to the target site for homologous replacement. Therefore, the triple helix site can be hundreds of base pairs away from the target site. An episomal assay for proof-of-principle study will be presented and discussed.
Gene Therapy 2002 9, 703-707. DOI: 10.1038/sj/gt/3301736
nature.com
Fast forward to the present ....
M Dutreix defines the Key problem with gene targeting
as ...
One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ
and goes on to describe her current research ....
1: Nucleic Acids Res 2003 Feb 1;31(3):1006-12 Related Articles, Links
Stimulation of D-loop formation by polypurine/polypyrimidine sequences.
Biet E, Sun JS, Dutreix M.
UMR 2027 CNRS-Institut Curie, section Recherche, Batiment 110, Centre Universitaire, 91405 Orsay, France.
Most of the approaches used to correct gene mutations in mammalian cells involve the targeting of short nucleotide molecules to homologous chromosomal sequences and the replacement of resident sequences via homologous recombination and mismatch repair. The limited efficiency and inconsistent reproducibility of these techniques are major constraints to their use in gene therapy. One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ. We investigated the effects of flanking sequences on homologous recombination by means of an in vitro assay of the efficiency of oligonucleotide targeting to its homologous sequence on a large duplex molecule in a reaction catalysed by the Escherichia coli RecA protein. We demonstrated that polypurine.polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides. This result was reproduced with various PPT sequences and oligonucleotides. The stimulatory effect was observed at loci as far as 4000 bp from the PPT. The formation of complexes between the oligonucleotide and the duplex molecule depended on the extent of sequence similarity between the two DNAs and the presence of the RecA protein. The stimulatory effect was inhibited by excess RecA and restored by adding heterologous DNA. We suggest that PPT sequences induce conformational changes in duplex DNA, leading to the aggregation of molecules, facilitating homology searches. We compared, in vivo, the efficiency of the oligonucleotide-mediated correction of a URA3 chromosomal mutation for sequences with and without a PPT sequence in the vicinity. Consistent with our in vitro results, the efficiency of correction was eight times higher in the presence of the PPT sequence.
PMID: 12560497 [PubMed - indexed for MEDLINE]
at the same time that the above paper comes out Kmiec
is releasing the following abstract .....
1: Biochemistry 2003 Mar 11;42(9):2643-55 Related Articles, Links
The synaptic complex of RecA protein participates in hybridization and inverse strand exchange reactions.
Gamper HB, Nulf CJ, Corey DR, Kmiec EB.
Division of Hematology/Oncology, University of Pennsylvania School of Medicine, BRB II/III Room 713, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. hbgamper@mail.med.upenn.edu
RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.
PMID: 12614159 [PubMed - in process]
ncbi.nlm.nih.gov
Summary:
Kmiec mentions this ....
This hybridization reaction ... might initiate recombination-dependent DNA replication if it occurs in vivo.
Dutreix mentions this .....
We demonstrated that polypurine.polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides.
John McCarthy |
