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Repair of DNA interstrand crosslinks may take place at the nuclear matrix
Author(s) -
Atanassov Boyko,
Gospodinov Anastas,
Stoimenov Ivaylo,
Mladenov Emil,
Russev George,
Tsaneva Irina,
Anachkova Boyka
Publication year - 2005
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20518
Subject(s) - plasmid , transfection , microbiology and biotechnology , homologous recombination , dna , nuclear matrix , dna repair , gene , green fluorescent protein , biology , chemistry , genetics , chromatin
Host cell reactivation assay using Trioxsalen‐crosslinked plasmid pEGFP‐N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP‐1, which did not contain the promoter of the egfp gene, and with pEGFP‐G − , which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%–2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP‐N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose‐dependant way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment. © 2005 Wiley‐Liss, Inc.