Increased activity and fidelity of DNA polymerase beta on single-nucleotide gapped DNA.
DNA polymerase beta (pol beta) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol beta activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol beta (kcat/Km,dNTPapp) is strongly influenced by gap size and the presence of a phosphate group at the 5'-margin of the gap. pol beta exhibited the highest catalytic efficiency on 5'-phosphorylated 1-nucleotide gapped DNA. This efficiency was >/=500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO4) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol beta, as judged by its ability to form G-N mispairs, was also higher (10-100 times) on 5'-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol beta is to fill 5'-phosphorylated 1-nucleotide gaps.
Chagovetz
A M
AM
Department of Biochemistry, Eccles Institute of Human Genetics and Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112, USA.
Sweasy
J B
JB
Preston
B D
BD
D000818:Animals; D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D004247:DNA; D019951:DNA Polymerase beta