The efficiency of hypoxanthine excision by alkyladenine DNA glycosylase is altered by changes in nearest neighbor bases.
Alkyladenine DNA glycosylase (AAG) excises a structurally diverse group of damaged purines including hypoxanthine, 1,N(6)-ethenoadenine, 3-methyladenine, and 7-methylguanine from DNA to initiate base excision repair at these sites. Excision occurs in an enzyme.DNA complex in which the damaged base is flipped out of the DNA helix into the enzyme active site. To determine whether local DNA sequence could affect the overall efficiency of excision of hypoxanthine from DNA, single-turnover kinetics of excision, AAG.DNA binding, and melting temperatures were measured for DNA substrates that differed in the base pairs immediately 5' and 3' to hypoxanthine. When Hx was flanked by a 5'G and a 3'C, the efficiency of excision was reduced dramatically in comparison to a duplex containing a 5'T and 3'A. The reduction in excision efficiency was largely due to a decrease in binding affinity of AAG for DNA. The overall effect of GC versus TA nearest neighbors was to magnify the difference in the efficiencies of excision of Hx from pairs with thymine and difluorotoluene from a factor of 5 to a factor of about 100. In general, DNA substrates that were more stable as indicated by higher melting temperatures gave reduced efficiencies of excision of Hx. These results are discussed in terms of a model in which the relative stabilities of base-flipped versus unflipped complexes contribute the overall efficiency of excision and substrate specificity of AAG.
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, 1600 SW Archer Road, JHMHC Room R3-234, Gainesville, FL 32610-0245, USA.
Aarthy C
AC
Vallur
Robyn L
RL
Maher
Linda B
LB
Bloom
D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D013379:Substrate Specificity; D044366:Transition Temperature
D019271:Hypoxanthine; D004247:DNA; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases