Pol ε poorly extends D-loops in comparison to Pol δ but is proficient to extend primed single-stranded DNA using the same substrates under the same conditions. ( A ) In vitro D-loop reactions using a 37-mer oligonucleotide were reconstituted using purified S. cerevisiae proteins as described in Materials and Methods. ( B ) Product analysis of reconstituted D-loop reactions containing either Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) at 0, 2 (not for Klenow), 5 and 10 min extension times. ( C ) Extension of primed single-stranded circular template DNA using a 37-mer oligonucleotide. ( D ) Product analysis of primer extension on denaturing gels of reaction containing Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) each plus or minus 10 nM PCNA/RFC at 0, 2 (not for Klenow), 5 and 10 min extension times. A 100 nt size ladder is shown in the left-most lane.
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