Kinetics of the synthesis of adducts between salmon testis DNA and platinum(II) compounds were measured by their effects on DNA synthesis, circular dichroism, and ethidium bromide dependent fluorescence. Transient incorporation of [14C]cyanide into DNA adducts of of cis-diammineaquochloroplatinum(II) and respectively cis-diamminediaquoplatinum(II) compounds but not of trans-diammineaquochlorplatinum(II) was observed. A minimal kinetic scheme is derived, in which a transient monodentate DNA-platinum(II) adduct is formed in a bimolecular reaction between DNA and aquated platinum(II) compounds. Second-order rate constants are 2000-3000 M-1 min-1 for cis-diamminediaquoplatinum(II) and 280-400 M-1 min-1 for cis- and trans-diammineaquochloroplatinum(II), respectively. The dependence of pseudo-first-order rate constants is not linear for high concentrations of DNA, suggesting competitive formation of more than one primary adduct. The monodentate adducts inhibit DNA polymerase catalyzed DNA synthesis. The biomolecular reaction is followed by a rearrangement (rate constant 0.22 min-1) that gives rise to most of the decrease in the fluorescence intensity and that depends on the state of aquation of the DNA-bound platinum(II) complex. By exchange of coordinated water with a second nucleotide, the monodentate adduct can form cross-links in a reaction joining the rearrangement. Adducts containing a chloro group liberate it by hydrolysis prior to cross-linking. In the case of the trans-platinum(II) adduct, the hydrolysis is aided by the trans effect of the bound first nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-a-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-a-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(viny1 sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (A') interspaced between them. Poly(L-malate) ( N = 5 ) and pOly(D,L-malate) ( N = 5 ) are the most efficient inhibitors, followed by poly(L-aspartate) ( N = 6), poly(acry1ate) ( N = 3), poly(Lglutamate) ( N = 8), poly(viny1 sulfate) ( N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-a-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-a-primase in the G 2 phase and M phase of the plasmodial cell cycle.Plasmodia1 cells of P. polycephalum have been of particular interest to cell biologists because of their giant multinucleated forms and their high synchrony in nuclear division [l]. In some instances, they resemble syncytically organized cells found during early stages of embryogenesis. P. polycephalum develops DNA polymerase a and newly replicated DNA may interact during the cell cycle competitively in such a way that DNA polymerase a is active during the S phase and inactive during the G2 phase. In this hypothesis, the initiation of histone synthesis marks the beginning of the S phase ([5], for a recent review about histones see [6]). The newly synthesized histones displace DNA polymerase CI from poly(L-malate) by competition. The released polymerase becomes involved in DNA replication until histone synthesis ceases, and histones are consumed in the energetically highly favorable formation of nucleosomes by newly replicated DNA. Free poly(L-malate) reassociates with DNA polymerase a at the onset of the G2 phase and thereby terminates DNA synthesis. Thus, poly(~-malate) may function in stock piling inactive DNA polymerase during the G 2 phase and M phase of the cell cycle. In the intact cell, DNA polymerase a is associated with DNA primase. Activities of these enzymes function coordinately during the start of DNA synthesis at replication origins and during initiation and elongation of Okazaki pieces (for a recent review see [...

No abstract

No abstract