Abstract
Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA polymerase (pol) beta, a family X polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol beta insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol beta bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.
Highlights
We analyze the kinetics of pol  insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant
Nontemplated DNA Polymerase  Insertion Specificity— Structural characterization of pol  bound to blunt-end DNA suggested that dATP could be preferentially added in a nontemplated reaction (31)
It was shown that dNTP selection/insertion “opposite” a synthetic abasic site in the context of surrounding single-stranded DNA was influenced by the identity of the adjacent downstream template base
Summary
Materials—Ultrapure deoxynucleoside triphosphates and MicroSpin G-25 columns were from GE Healthcare. Primer, and downstream oligonucleotides was mixed in a 1:1:1 ratio and annealed using a PCR thermocycler by heating for 10 min at 90 °C and cooling to 4 °C (1 °C/min) resulting in a 1 mM mixture of gapped duplex DNA. This solution was mixed with an equal volume of pol  at 4 °C, and the mixture was warmed to 35 °C and gradually cooled to 4 °C.
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