The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells.
Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.
Brooks
P J
PJ
Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892, USA. pjbrooks@niaaa.nih.gov
Wise
D S
DS
Berry
D A
DA
Kosmoski
J V
JV
Smerdon
M J
MJ
Somers
R L
RL
Mackie
H
H
Spoonde
A Y
AY
Ackerman
E J
EJ
Coleman
K
K
Tarone
R E
RE
Robbins
J H
JH
D000328:Adult; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D003839:Deoxyadenosines; D005786:Gene Expression Regulation; D006801:Humans; D018384:Oxidative Stress; D051381:Rats; D014983:Xeroderma Pigmentosum
D003839:Deoxyadenosines; C056752:8,5'-cyclo-2'-deoxyadenosine