Host cell reactivation by human cells of DNA expression vectors damaged by ultraviolet radiation or by acid-heat treatment

Abstract

We utilized a plasmid vector host cell reactivation assay to probe the biological functioning of DNA expression vectors and their encoded genes. We studied the effect of ultraviolet radiation or acid-heat treatment on the transient expression of genes transfected into normal human cells and into DNA repair deficient (xeroderma pigmentosum) cells and modification of gene expression by sodium butyrate. U.v. inactivation of transient expression of the bacterial gpt gene contained in a non-replicating expression vector plasmid, pSV2catSVgpt, was much greater in three xeroderma pigmentosum lines than in the four other human cell lines tested. In contrast, treatment of pSV2catSVgpt with acid and heat to produce apurinic sites resulted in a similar slope of the inactivation curve of the bacterial cat gene in the repair deficient and repair proficient cells. Thus, u.v. damage of DNA expression vectors was subject to repair by the normal host cells, but acid-heat treatment resulted in damage (apurinic sites) that was handled in a similar manner by excision repair deficient and excision repair proficient human cells. In both normal and xeroderma pigmentosum cells sodium butyrate treatment of cells resulted in a greater stimulation of chloramphenicol acetyltransferase expression with u.v. damaged than with undamaged plasmid. This assay thus permits examination of the effects of defined types of DNA damage on plasmid expression and study of its modulation by cellular repair activities.

Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

“…The CAT reporter gene has been used extensively to monitor ER in situ for cultured cells (25,41,49,50,68). UV-irradiated aliquots of plasmid pSVCATgpt were introduced into UV135 and 38.4.4 cells and the cDNA-cosmid transformants as described in Materials and Methods.…”

Analysis of DNA Repair Using Transfection-Based Host Cell Reactivation

“…BER is also known to remediate some of the damage caused by exposure of DNA to oxidative agents, such as ozone and sources of singlet oxygen (25,26). The steps in BER beyond the initial glycosylation can be analyzed by acid/heat treatment of the plasmid to produce apurinic sites (27), or apurinic sites can be specifically introduced (28). A specific modification of the HCR assay has been developed to allow for analysis of DNA mismatch repair via generation of microsatellite instability following exposure of the reporter plasmid to etoposide or fotemustine (29).…”

“…BER is also known to remediate some of the damage caused by exposure of DNA to oxidative agents, such as ozone and sources of singlet oxygen (25,26). The steps in BER beyond the initial glycosylation can be analyzed by acid/heat treatment of the plasmid to produce apurinic sites (27), or apurinic sites can be specifically introduced (28).…”

Organization of Plasmid DNA into Nucleosome-Like Structures after Transfection in Eukaryotic Cells

“…In this assay, the transcriptional activity of the reporter gene reactivated in the host cell as a result of DNA repair is monitored at different times after transfection (1). initially viral DnA vectors were used but later plasmid HCR assays have been described, which monitor cellular repair by measuring the transient expression of enzymatic marker genes (7,16). the hcR assay was originally developed with the plasmid pCMVcat harboring the chloramphenicol acetyltransferase reporter gene (1), and it has also been successfully modified using a recombinant luciferase reporter plasmid pCMVluc (17).…”