Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork.

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Abstract
At an early purification stage, DNA polymerase .alpha. holoenzyme from calf thymus can be separated into 4 different forms by chromatography on DEAE-cellulose. All 4 enzyme forms (termed A, B, C and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase .alpha., a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3''-5'' exonuclease and RNase H activity. Peaks B, C and D all contain, together with DNA polymerase .alpha., activities of primase and DNA topoisomerase type II. Peak B is enriched in an RNase H, and peaks C and D are enriched in a 3''-5'' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the 4 peaks gave evidence of unexpectedly large forms of DNA polymerase .alpha. (> 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase .alpha.. Peak C is more resistant to inhibition by salt and spermidine than the other 3 enzyme forms. The existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C and D) is suggested. The salt-resistant C form might represent a functional DNA polymerase .alpha. holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.