DNA polymerase δ and ɛ holoenzymes from calf thymus

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Abstract
Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) δ completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (orE.coli singlestrand DNA binding protein, SSB). Pol ɛ core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol ɛ was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol ɛ by PCNA/RF-C/SSB appears to be the superposition of two events: pol, ɛ holoenzyme (pol ɛ, PCNA, RF-C) synthesized longer products than its pol ɛ core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol α/primase with pol δ or pol ɛ holoenzymes on unprimed M13 DNA. While pol δ displayed higher dNMP incorporation than pol ɛ, when a single primer was preannealed to DNA, pol ɛ was more efficient in the utilization of the primers synthesized by pol α/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol ɛ holoenzyme, than for pol δ. Our data support the hypothesis of pol δ as the leading and pol ɛ as the second lagging strand replication enzyme.