Maize replicative α‐type DNA polymerase: separation of polymerase and primase activities and recognition of primase subunits

Abstract

DNA polymerase and DNA primase activities in the maize alpha-type DNA polymerase 2 were dissociated and DNA polymerase-free DNA primase was studied. DNA primase synthesized primers that were 8-34 nucleotides long, with more intense bands at 15-17 nucleotides in length. DNA polymerase 1 (a putative delta-type enzyme) or DNA polymerase 2 were assayed after template-priming with purified DNA primase and showed a differential use of templates: whereas DNA polymerase 2 used a polydT template more efficiently than a natural template, DNA polymerase 1 used both of them poorly. The molecular size of DNA primase was estimated to be 68 kDa by gel filtration, western blotting and by a DNA primase 'trapping' assay.

Expression of a maize δ‐type DNA polymerase during seed germination

“…This was prepared as described before (García et al 2002); briefly, 1 g of maize embryo axes was homogenized with 3 ml of buffer A [Tris–HCl pH 7.6, 50 m M , KCl 25 m M , MgCl 2 1 m M , potassium phosphate pH 7.6, 30 m M , 2‐mercaptoethanol 1 m M , glycerol 20% and a tablet of protease inhibitors (Complete, Roche)] per 50 ml of buffer. The homogenate was centrifuged at 12 000 g for 15 min, the supernatant was centrifuged at 100 000 g for 2.5 h, nucleic acids were precipitated by adding 10% protamine, 10 µl per ml homogenate for 30 min, the solution was centrifuged at 15 000 g for 30 min and the supernatant was stored at 4°C for further studies.…”

“…Resin (50 µl) containing the antibody was incubated with 500 µg of crude protein extract from maize embryo axes, previously imbibed for different germination periods, for 12 h at 4°C and then centrifuged at 30 g for 1 min; the resin was washed twice with PBS, and the supernatant was discarded. This resin, containing bound Zmpolδ, was used to measure DNA pol activity; for this, 200 µl of reaction mixture for DNA pol activity (García et al 2002) was added to the resin, the mixture was incubated for 1 h at 37°C and then the supernatant was recovered, receiving 1 ml TCA 10% and was filtered through Whatman GF‐C filter papers. Radioactivity in the filtrates was measured using a scintillation counter.…”

“…DNA pol 1 has a native molecular mass of 350 kDa, prefers poly‐dA/oligo dT to activated DNA or poly‐rA/oligo dT and its activity is stimulated by maize PCNA (García et al 1997). DNA pol 2 has a native molecular mass around 400 kDa, its activity increases importantly during maize germination (Coello et al 1992), appears to be regulated by phosphorylation and it is a low processivity enzyme (Coello and Vázquez‐Ramos 1995a, 1995b) and contains a strongly associated DNA primase activity (García et al 2002).…”

Genome-Wide Analysis of the Core DNA Replication Machinery in the Higher Plants Arabidopsis and Rice

“…1C; The POLA complex includes a catalytic subunit (POLA1), two primase subunits (POLA3 and POLA4), and POLA2, which is thought to tether the complex to the replication fork (Frick and Richardson, 2001). Protein complexes containing polymerase and primase activity have been purified from a variety of plant systems (Coello and Vazquez-Ramos, 1995;Garcia et al, 2002), demonstrating that a POLA-like function exists in plants. However, sequence homology has been investigated only for the POLA1 subunit in rice (Yokoi et al, 1997).…”