Catalytic center of DNA polymerase beta for excision of deoxyribose phosphate groups.
The amino-terminal 8-kDa domain of vertebrate DNA polymerase beta (pol beta) has an activity to excise deoxyribose phosphate (dRP) groups from 5'-incised apurinic/apyrimidinic (AP) sites during base excision repair. The excision reaction proceeds via a beta-elimination reaction following formation of a Schiff base between an aldehyde group of the AP site and an amino group of the enzyme. Here we report that the Lys-72 residue of this enzyme is the catalytic center for dRP excision. Substitutions of Lys-72 with Arg or Gln reduced the dRP excision activity to less than 1% of the wild-type 8-kDa domain, while substitutions of Lys-35, Lys-68, or Lys-84 did not abolish its activity. The Lys-72 mutations also significantly decreased Schiff base intermediates trapped by reduction with sodium borohydride. The 8-kDa domain alone was able to bind preferentially to a single-nucleotide gap or 5'-incised synthetic AP site on double-stranded DNA. The Lys-72 mutations did not affect this damage-specific DNA binding activity. When introduced into the intact enzyme, a mutation of Lys-72 to Arg did not affect DNA synthesis activity of pol beta, but eliminated the repair activity. Addition of the wild-type 8-kDa domain to this reaction restored the repair activity. These results indicate a specific role of Lys-72 of pol beta in the dRP excision during base excision repair.
Matsumoto
Y
Y
Department of Radiation Oncology and Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Kim
K
K
Katz
D S
DS
Feng
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015374:Biosensing Techniques; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012267:Ribosemonophosphates / Q000737:chemistry* / Q000378:metabolism
D012267:Ribosemonophosphates; D004247:DNA; D019951:DNA Polymerase beta