New immunoaffinity-LC-MS/MS methodology reveals that Aag null mice are deficient in their ability to clear 1,N6-etheno-deoxyadenosine DNA lesions from lung and liver in vivo.
The mouse alkyladenine DNA glycosylase (Aag) initiates base excision repair with a broad substrate range that includes the highly mutagenic exocyclic etheno DNA base adduct 1,N6-ethenodeoxyadenosine ((epsilon)dA). Previous attempts to determine the in vivo role of Aag in (epsilon)dA repair were complicated by technological difficulties in measuring low levels of (epsilon)dA in genomic DNA. Here we describe the development of a new method for (epsilon)dA detection in genomic DNA that couples an immunoaffinity purification with LC-MS/MS analysis and that utilizes an isotopically labeled internal standard. We go on to describe the application of this method to measuring the in vivo repair of (epsilon)dA base lesions in liver and lung tissue of wild type and Aag null mice. Our results demonstrate that while Aag clearly represents the major DNA repair enzyme for the in vivo removal (epsilon)dA bases, these lesions can also be eliminated from the genome via an alternative mechanism.
Ham
Amy-Joan L
AJ
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill 27599, USA.
Engelward
Bevin P
BP
Koc
Hasan
H
Sangaiah
Ramiah
R
Meira
Lisiane B
LB
Samson
Leona D
LD
Swenberg
James A
JA
D000818:Animals; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods*; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D003839:Deoxyadenosines / Q000378:metabolism*; D008099:Liver / Q000473:pathology; D008168:Lung / Q000473:pathology; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D018345:Mice, Knockout
C115182:1,N(6)-etheno-2'-deoxyadenosine; D003839:Deoxyadenosines; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases