Repair of DNA interstrand crosslinks may take place at the nuclear matrix

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 e...

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Bibliographic Details
Published in:Journal of cellular biochemistry 2005-09, Vol.96 (1), p.126-136
Main Authors: Atanassov, Boyko, Gospodinov, Anastas, Stoimenov, Ivaylo, Mladenov, Emil, Russev, George, Tsaneva, Irina, Anachkova, Boyka
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cell Line, Tumor
Cricetinae
DNA - drug effects
DNA - metabolism
DNA recombination
DNA repair
DNA Repair - physiology
Genes, Reporter
host cell reactivation
Humans
interstrand crosslinks
nuclear matrix
Nuclear Matrix - enzymology
Nuclear Matrix - physiology
Plasmids
Transfection
Trioxsalen - pharmacology
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Summary: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.
ISSN:0730-2312
1097-4644
DOI:10.1002/jcb.20518