DNA polymerase delta requires proliferating cell nuclear antigen and replication factor C to form a holoenzyme efficient in DNA synthesis. We have analyzed three different aspects of calf thymus DNA polymerase delta holoenzyme: (i) analysis of pausing during DNA synthesis, (ii) replication of double-stranded DNA in the absence of additional factors, and (iii) replication of double-stranded DNA in the presence of the two known replicative DNA helicases from simian virus 40 and bovine papilloma virus. DNA polymerase delta holoenzyme replicated primed single-stranded DNA at a rate of 100-300 nucleotides/min, partially overcoming multiple pause sites on DNA. While Escherichia coli single-strand DNA binding protein helped DNA polymerase delta pass through pause sites, the DNA polymerase delta itself appeared to dissociate from the template in the absence of synthesis or when encountering pause sites. Proliferating cell nuclear antigen likely remained on the template. DNA polymerase delta holoenzyme could perform limited strand displacement synthesis on double-stranded gapped circular DNA, and this reaction was not stimulated either by replication protein A or by E. coli single-strand DNA binding protein. DNA polymerase delta holoenzyme could efficiently cooperate with replicative DNA helicases from simian virus 40 (large T antigen) and bovine papilloma virus 1 (protein E1) in replication through double-stranded DNA in a reaction that required replication protein A or E. coli single-strand DNA binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)