Abstract
The fidelity of Schizosaccharomyces pombe DNA polymerase delta was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol delta alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol delta holoenzyme (HE) (pol delta + PCNA + RFC) was 4.6 x 10(-4) for T.G mispairs, 5.3 x 10(-5) for G.G mispairs, and 4.5 x 10(-6) for A.G mispairs. The T.G misincorporation frequency for pol delta without the accessory proteins was unchanged. The fidelity of pol delta HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol delta HE with E. coli pol IIIalpha HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.
Highlights
The enzymes principally responsible for catalyzing procaryotic and eucaryotic DNA replication share many common elements
Extensive studies on the fidelity properties of core DNA polymerases have been reported over the past 3 decades focusing on biochemical and kinetic analysis of deoxynucleotide insertion specificity and the reduction in pol-generated errors by proofreading exonucleases (15–18), whereas there are but a paucity of experiments reporting on the fidelity properties of the more biologically relevant pol HE systems
Previous experiments employing pol HE systems have attempted to probe the fidelity of leading versus lagging strand synthesis using mutational reporter sequences (19, 20) and to visualize synthesis past DNA damage sites using two-dimensional gel electrophoresis (21, 22)
Summary
Proteins cloned S. pombe pol d, PCNA, and RFC were purified as described (30). The enzyme reaction buffer contained 40 mM TriszHCl, pH 7.8, 170 mg/ml bovine serum albumin, 0.5 mM dithiothreitol, and 7 mM MgCl2. DNA-binding protein; p/t DNA, primer-template DNA; nt, nucleotide; ssDNA, single-stranded DNA. DNA-binding protein and bovine serum albumin were purchased from Amersham Pharmacia Biotech. The 30-mer primer was annealed at the middle of the template leaving equal length (35 nt) ssDNA overhangs on each side. The matched 35-mer primer was annealed to the template leaving 35 nt of ssDNA at the 39-end of the template and 30 nt of ssDNA at 59-end. The mismatched 35-mer primer was identical to the matched 35-mer except that the nucleotide at the 39-end contained an A in place of C. Nucleotides—dNTP substrates were purchased from Amersham Pharmacia Biotech. Nucleotides—dNTP substrates were purchased from Amersham Pharmacia Biotech. [g-32P]ATP (4500 Ci/mmol) was purchased from ICN Radiochemicals
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