Recognition of oxidized thymine base on the single-stranded DNA by replication protein A.
Replication protein A (RAP) is a eukaryotic single-stranded DNA binding protein involved in DNA replication, repair, and recombination. Recent studies indicate that RPA preferentially binds the damaged sites rather than the undamaged sites. Therefore, RPA is thought to be a member ofrepair factories or a sensor of lesion on DNA. To obtain further information of behavior of RPA against the oxidized lesion, we studied the binding affinity of RPA for the single-stranded DNA containing 5-formyluracil, a major lesion of thymine base yielded by the oxidation, using several synthetic oligonucleotides. The affinity of RPA for oligonucleotides was determined by gel shift assay. Results suggest that the surrounding sequence of 5-formyluracil may affect the affinity for RPA, and that the 5-formyluracil on the purine stretch but not the pyrimidine stretch increases the affinity for RPA. Results of affinity labeling experiment of RPA with the oligonucleotides containing 5-formyluracil indicate that RPA1 subunit may directly recognize and bind to the 5-formyluracil on the single-stranded DNA.
Irie
Daisuke
D
Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan.
Ono
Akira
A
Izuta
Shunji
S
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry*; D010084:Oxidation-Reduction; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling; D013941:Thymine / Q000737:chemistry*; D014498:Uracil / Q000031:analogs & derivatives / Q000737:chemistry; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D004277:DNA, Single-Stranded; D051716:Replication Protein A; D029867:Xenopus Proteins; C007777:5-formyluracil; D014498:Uracil; D013941:Thymine