The high binding affinity of human ribosomal protein S3 to 7,8-dihydro-8-oxoguanine is abrogated by a single amino acid change.
Previous studies have shown that human ribosomal protein S3 (hS3) has a high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) residues in DNA and interacts with the human base excision repair (BER) proteins OGG1 and APE/Ref-1. We used a combination of computational and experimental approaches to understand the role of hS3 in BER and its potential to hinder repair of 8-oxoG lesions by OGG1 and APE/Ref-1. Sequence analysis was employed to identify hS3 residues likely to be involved in binding to 8-oxoG. One putative site, lysine 132 (K132), located in a helix-hairpin-helix DNA binding motif, was mutated to alanine (K132A). The hS3-K132A mutant retained the ability to cleave abasic DNA, but its capacity to bind 8-oxoG was abrogated completely. The ability of OGG1 to cleave an 8-oxoG-oligonucleotide substrate pre-incubated with hS3 or hS3-K132A was also tested. Pre-incubations with wild-type hS3 and 8-oxoG-containing oligonucleotides completely prevented the subsequent removal of 8-oxoG by OGG1. On the other hand, OGG1 incubations combined with hS3-K132A stimulated cleavage of 8-oxoG in excess of two-fold, confirming previous observations that hS3 positively interacts with OGG1, but only under conditions in which the binding of hS3 to 8-oxoG is limited. Overall, the ability of OGG1 to repair 8-oxoG is compromised when hS3 is bound to 8-oxoG sites. Conversely, in the absence of DNA binding, hS3 interacts positively with OGG1 to produce a more robust removal of 8-oxoG residues in DNA.
Hegde
Vijay
V
Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, 70808, USA.
Wang
Mu
M
Mian
I Saira
IS
Spyres
Lea
L
Deutsch
Walter A
WA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045647:DNA Glycosylases / Q000378:metabolism; D004260:DNA Repair; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C044152:ribosomal protein S3; D006151:Guanosine; C046215:8-hydroxyguanosine; D004247:DNA; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human