The DNA-polymerase inhibiting activity of POIY(β-L-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum

The naturally synchronous plasmodia of myxomycetes synthesize pply(beta-L-malic acid), which carries out cell-specific functions. In Physarum polycephalum, poly(beta-L-malate) [the salt form of poly(beta-L-malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell-cycle-dependent, purified DNA polymerase alpha of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [H-3]thymidine 5'-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(beta-L-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(beta-L-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol 70, 373-380]. To explain the variation in the cell cycle, a periodic competition for poly(beta-L-malate) between DNA polymerases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competition they displace DNA polymerase from the complex of poly(beta-L-malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(beta-L-malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.