TY - JOUR
T1 - Crosslinking of DNA and proteins induced by protein hydroperoxides
AU - Gebicki, Silvia
AU - Gebicki, Janusz M.
PY - 1999/3/15
Y1 - 1999/3/15
N2 - Exposure of DNA to several proteins peroxidized by radiation-generated hydroxyl free radicals resulted in formation of crosslinks between the macromolecules, detected by retardation and broadening of DNA bands in agarose gels. This technique proved suitable for the study of crosslinking of DNA with peroxidized BSA, insulin, apotransferrin and α casein, but not with several other proteins, including histones. The crosslinking depended on the presence of intact hydroperoxide groups on the protein, on their number, and on the duration of the interaction with DNA. All DNA samples tested, pBR322, pGEM, λ/HindIII and pUC18, formed crosslinks with the peroxidized BSA. Sodium chloride and formate prevented the crosslinking if present during incubation of the peroxidized protein and DNA, but had no effect once the crosslinks had formed. The gel shift of the crosslinked DNA was reversed by proteolysis, indicating that the DNA mobility change was due to attachment of protein and that the crosslinking did not induce DNA strand breaks. The metal chelators Desferal and neocuproine reduced the extent of the crosslinking, but did not prevent it. Scavengers of free radicals did not inhibit the crosslink formation. The DNA-protein complex was not disrupted by vigorous agitation, by filtration or by non-ionic detergents. These observations show that the crosslinking of DNA with proteins mediated by protein hydroperoxides is spontaneous and probably covalent, and that it may be assisted by transition metals. It is suggested that formation of such crosslinks in living organisms could account for some of the well-documented forms of biological damage induced by reactive oxygen species-induced oxidative stress.
AB - Exposure of DNA to several proteins peroxidized by radiation-generated hydroxyl free radicals resulted in formation of crosslinks between the macromolecules, detected by retardation and broadening of DNA bands in agarose gels. This technique proved suitable for the study of crosslinking of DNA with peroxidized BSA, insulin, apotransferrin and α casein, but not with several other proteins, including histones. The crosslinking depended on the presence of intact hydroperoxide groups on the protein, on their number, and on the duration of the interaction with DNA. All DNA samples tested, pBR322, pGEM, λ/HindIII and pUC18, formed crosslinks with the peroxidized BSA. Sodium chloride and formate prevented the crosslinking if present during incubation of the peroxidized protein and DNA, but had no effect once the crosslinks had formed. The gel shift of the crosslinked DNA was reversed by proteolysis, indicating that the DNA mobility change was due to attachment of protein and that the crosslinking did not induce DNA strand breaks. The metal chelators Desferal and neocuproine reduced the extent of the crosslinking, but did not prevent it. Scavengers of free radicals did not inhibit the crosslink formation. The DNA-protein complex was not disrupted by vigorous agitation, by filtration or by non-ionic detergents. These observations show that the crosslinking of DNA with proteins mediated by protein hydroperoxides is spontaneous and probably covalent, and that it may be assisted by transition metals. It is suggested that formation of such crosslinks in living organisms could account for some of the well-documented forms of biological damage induced by reactive oxygen species-induced oxidative stress.
KW - DNA gel shift
KW - free radical
KW - oxidation
KW - radiation
KW - reactive oxygen species
UR - http://www.scopus.com/inward/record.url?scp=0033560051&partnerID=8YFLogxK
U2 - 10.1042/0264-6021:3380629
DO - 10.1042/0264-6021:3380629
M3 - Article
C2 - 10051432
AN - SCOPUS:0033560051
SN - 0264-6021
VL - 338
SP - 629
EP - 636
JO - Biochemical Journal
JF - Biochemical Journal
IS - 3
ER -