Abstract
Quantitative rates of DNA synthesis can be determined by DNA:propidium fluorescence measurements of synchronized cells progressing through S-phase. We have previously reported that HeLa cells have discontinuous rates with values of about 2.9, 1.6, and 4.4 pg of DNA/h for early, middle, and late S-phase, respectively. In attempts to understand why two peaks of DNA synthesis rates are observed, we have examined the nuclear DNA polymerases alpha and beta over the S-phase. Nuclear matrices isolated from HeLa cells contained 2% of the alpha polymerase and 12% of the beta polymerase that was present in cell lysates, and about 2% of the original DNA. The amounts of endogenous DNA synthesis in isolated nuclear matrices were comparable to the amounts observed when exogenous DNA was added. DNase treatment abolished the endogenous DNA synthesis but not the exogenous DNA synthesis, suggesting that polymerase alpha binding does not depend on matrix-bound DNA. As synchronized cells progressed through the S-phase, there appeared two peaks of enzymatic activity of alpha polymerase bound to the nuclear matrix which correlated with in vitro DNA synthesis in these nuclear matrices and with the two peaks of quantitative DNA synthesis rates. Two peaks of alpha polymerase activity were also observed with isolated nuclei, but not with cell lysates or cytosol. Our results suggest that, over the S-phase, the differential binding of polymerase alpha to the nuclear matrix determines the differential rates of DNA synthesis.
Highlights
The Interrelation between DNA Synthesis Rates and DNA Polymerases Bound to theNuclear Matrix inSynchronized HeLa Cells”
An increase in thepol’ (Y activity of isolated nuclei was observed between the GI-phaseand the mid S-phase (Chiu and Baril, 1975).We have demonstrated that quantitative ratesof DNA synthesis, expressed as pg of DNA synthesized per hr, are far more reliable than estimates based on incorporation of [3H] dThd; thismethod reflects the numbers of cells in S-phaseas well as thymidine uptake, thymidine pools, and conversion into TTP (Collins, 1978).For example, the quantitative rates added
Gressed through theS-phase, there appeared two peaks The nuclear matrix is experimentally defined as theresidual of enzymatic activity of a polymerase bound to the nuclear matrix which correlated with in vitro DNA synthesis in these nuclear matrices and with the two peaks of quantitative DNA synthesis rates.Two peaks of a polymerase activity were observed with isolated nuclei, but not with cell lysates or cytosol
Summary
@ 1985by The American Societyof Biological Chemists, Inc. Vol 260, No 7, Issue of April 10, pp. 4229-4235,1985 Printed in U.S.A. The amounts of endogenous DNA synthesis in isolated nuclear matrices werecomparable to the amounts observed when exogenous DNA was synchronized HeLa cells, only two and three time points of the S-phase were examined by Spadari andWeissbach (1974) and Chiu and Baril (1975), respectively. (1980) demonstrated in an SV40 system that polymerase vecz et al (1975) has led to the terms“early” and “late”DNA a was tightly associated with SV40 chromatin, and Smith and (Holmquist et al, 1982; Goldman et al, 1984) It has been Berezney (1983) demonstrated that substantial amounts of suggested that early and late DNA may represent two func- polymerase a (but not polymerase p), were tightly bound to tionally distinct domains in the nucleus and that theperiod the proteinacious nuclear matrix during DNA replication. DNA SynRtahteessis and Polymerases in HeLa possibility of a relationship between binding of nuclear DNA polymerases to the nuclear matrixand the rates of DNA synthesis
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