Recombinant Phabs reactive with 7,8-dihydro-8-oxoguanine, a major oxidative DNA lesion.
Antibody Fabs that bind to DNA damages provide useful models for understanding DNA damage-specific protein interactions. BSA and RSA conjugates of the nucleoside and nucleotide derivatives of the oxidative DNA lesions, 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), were used to immunize mice. RNA from the responders was isolated and used to repertoire clone and phage display Fabs that bind to these haptens. Direct binding and competitive enzyme-linked immunosorbent assay (ELISA) demonstrated that phage Fabs (Phabs) specific for 8-oxopurine-BSA conjugates and 8-oxoguanine were produced although the Phabs did not react with 8-oxopurines in DNA. Amino acid sequence comparisons among clones having different binding properties suggested that a relatively small portion of the binding surfaces defined by the complementarity determining regions (CDR) accounted for hapten binding specificity, whereas other regions appeared to stabilize hapten binding by interacting with protein or DNA epitopes. Chain shuffling between 8-oxopurine-BSA binding Fabs and a DNA binding Fab showed that the heavy chain of the DNA binder conferred DNA binding capacity to the light chain of only one of the 8-oxopurine-BSA binders. Homology modeling of the 8-oxoG-specific clone g37 showed significant similarities to two previously isolated monoclonal antibodies specific for single-stranded nucleic acids. In the 8-oxoG Fab, which did not bind to DNA, the presumptive DNA binding canyon was blocked by heavy chain residues in the CDR2 region and appeared to lack part of the canyon wall due to the different placement of the light chain framework region.
Bespalov
I A
IA
Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Burlington 05405, USA.
Purmal
A A
AA
Glackin
M P
MP
Wallace
S S
SS
Melamede
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA