Abstract
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.
Highlights
DNA polymerase  is an error-prone polymerase that plays a central role in mammalian base excision repair
DNA polymerase 1 plays a central role in mammalian base excision repair (BER [1,2,3,4,5]). pol  is a monomeric 39-kDa enzyme organized into a carboxyl-terminal 31-kDa domain that includes the polymerase active site and an aminoterminal 8-kDa domain that participates in DNA binding and harbors 5Ј-deoxyribose phosphodiesterase activity [6, 7]
Processive DNA synthesis on short (2– 6-nt) gaps is consistent with roles for pol  in long-patch BER [4] and in the completion of gap-filling synthesis initiated by other cellular DNA polymerases (9, 10, 14 –16)
Summary
DNA polymerase  (pol ) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the parameters governing pol  activity on 1-nt gapped DNA, we examined the steady-state kinetics of DNA polymerization on synthetic primer-templates of different structure. We show that the catalytic efficiency (kcat/ Km ap,pdNTP) and nucleotide insertion fidelity of pol  are strongly influenced by gap size and that the 5Ј-phosphorylation requirement is retained for these activities even on 1-nt gapped DNA.
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