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 character ize the mechanisms governing rat pol beta activity, we examined polyme rization on synthetic primer-templates of different structure. Steady- state kinetic analyses revealed that the catalytic efficiency of pol b eta (k(cat)/K-m,dNTP(app)) is strongly influenced by gap size and the presence of a phosphate group at the 5'-margin of the gap. pol beta ex hibited the highest catalytic efficiency on 5'-phosphorylated 1-nucleo tide gapped DNA. This efficiency was greater than or equal to 500 time s higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (wit h or without PO4) gapped DNAs and 2,500 times higher than on primer-te mplate 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 t imes) on 5'-phosphorylated single nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary fu nction of mammalian pol beta is to fill 5'-phosphorylated 1-nucleotide gaps.