DNA polymerase ? of Physarum polycephalum

Abstract

DNA polymerase delta from the phylogenetically ancient slime mold Physarum polycephalum has been 380-fold enriched from amoebae. It was found to have the properties typical for this type of DNA polymerase from higher eukaryotes with regard to effectors, template-primer acceptance, co-purification with 3'-5'-exonuclease activity, as well as the effect of endogenous proliferating cell nuclear antigen (PCNA) from amoebae on the stimulation and processivity of DNA synthesis. An identified cDNA fragment shows 65.5% identical amino acides with DNA polymerase delta from Saccharomyces pombe. The molecular mass of the polymerase is 125 kDa while that of PCNA is 35 kDa. During size-exclusion chromatography, the highly purified polymerase eluted in the position of 125 kDa, suggesting that no other proteins were tightly complexed with the enzyme. The DNA polymerases from the (mononucleate) amoebae and from the (multinucleate) plasmodia of P. polycephalum have very similar properties in contrast to their differences in phenotype and their mode of nuclear division. The polymerase shows a higher degree of similarity than DNA polymerase alpha, and especially the beta-like DNA polymerase, with the corresponding polymerases of higher eukaryotes. According to antibody staining, DNA polymerase delta is readily fragmented by proteases, even in the presence of inhibitor cocktails. Including freshly prepared cell lysates, proteolytic fragments are reproducible, the most abundant being 50 kDa in size. The DNA polymerase is recognized by the antisera against two peptides which have been derived by PCR-screening of plasmodial cDNA. One of the proteolytic splitting sites is located within an eight amino-acid stretch between the two antigenic sequences.

Molecular constituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoaffinity labelling

“…Rabbit antiserum against P . polycephalum DNA polymerases a and E (both as holoenzymes) and rabbit antiserum against peptides of P. polycephalum DNA polymerase 6 have been described (Achhammer et al, 1992(Achhammer et al, , 1995. Chicken antiserum against human PCNA was a gift from Dr B. Stillman (Cold Spring Harbor, USA).…”

“…The DNA polymerase a 140 kDa subunit was proteolytically unstable, forming an inactive 110 kDa fragment (Weber et al, 1988). The polymerase was found to differ in several respects from polymerases of yeasts and higher eukaryotes (Achhammer et al, 1995).…”

“…Its heavy immunostaining and photolabelling indicate that the 46 kDa PCNA-like species was very abundant and probably significant. We have recently purified and characterized a 35 kDa PCNA in P. polycephalum (Achhammer et al, 1995). It is now assumed that this form of PCNA is the product of partial proteolysis.…”

“…This polyanion is specifically synthesized in plasmodia but not in uninuclear cell forms of P. polycephalum. Its mode of action has been tentatively attributed to transport and storage of histones, DNA polymerases a and 6, and other nuclear proteins in DNA replication (Fischer et al, 1989;Holler et al, 1992b;Achhammer et al, 1995;. P-like DNA polymerase, which is probably not active in DNA replication, does not bind to PMLA.…”

“…To gain insight into evolutionary variability and synchrony maintenance, we began to characterize the replication apparatus of P. polycephafum. DNA polymerase u-primase complex (140 kDa polymerizing subunit, two primase subunits of 53 and 58 kDa, and a 82 kDa polypeptide) (Weber et al, 1988;Achhammer et al, 1992;, DNA polymerase 6 (125 kDa) and PCNA (35 kDa) (Achhammer et al, 1995) have been identified and characterized. The DNA polymerase a 140 kDa subunit was proteolytically unstable, forming an inactive 110 kDa fragment (Weber et al, 1988).…”