Date of Award


Degree Type


Degree Name

Doctor of Philosophy


This work is an in vitro study of the DNA polymerase {dollar}\alpha{dollar}-DNA primase complex, which is thought to synthesize DNA on the lagging strand at replication forks. We have purified the complex, and studied two aspects of RNA primed DNA synthesis catalyzed by this enzyme complex in vitro.;DNA polymerase {dollar}\alpha{dollar} has been purified from cultured mouse Ehrlich ascites tumour cells, by classical purification methods, to a specific activity of 60 000 units/mg total protein. The purified DNA polymerase {dollar}\alpha{dollar} complex has four subunits, 182, 70, 55, and 47kDa. A 3{dollar}\sp\prime\to 5\sp\prime{dollar} exonuclease activity, detected after treatment of the complex with ethylene glycol, cosedimented with the DNA polymerase activity, on sedimentation through glycerol gradients. The sensitivity of DNA polymerase {dollar}\alpha{dollar} to the nucleotide analogues butylphenyl-dGTP and butylanilino-dATP did not change when the 3{dollar}\sp\prime\to 5\sp\prime{dollar} exonuclease was active.;The effect of template DNA sequence on the rate of initiation of RNA primed DNA synthesis by the purified mouse DNA polymerase {dollar}\alpha{dollar}-primase complex was examined. We have observed discontinuous DNA synthesis, which was dependent on ATP or GTP. RNA primed initiation is favoured 10 nucleotides upstream of 3{dollar}\sp\prime{dollar} CCA and 3{dollar}\sp\prime{dollar} CCC sequences, which we have called {dollar}\psi{dollar}. Alteration of the {dollar}\psi{dollar} domain led to changes in the rate of RNA primed DNA synthesis, and kinetic analysis showed that these changes were due to less efficient interaction with the DNA template. Changes in the K{dollar}\sb{lcub}\rm m{rcub}{dollar} with respect to DNA concentration (from an optimal 6pM to 240pM) were observed when {dollar}\psi{dollar} was varied from the consensus sequences. Mutations in actual sites of priming led to an altered rate of synthesis of RNA primed DNA chains, reflected in changes in V{dollar}\sb{lcub}\rm max{rcub}{dollar} for the reaction (from 5.5-30pmol deoxynucleotides incorporated/hour).;DNA polymerase {dollar}\alpha{dollar} was also studied in a direct gap filling assay. DNA polymerase {dollar}\alpha{dollar} is able to fill gaps to completion. However, incorporation of the final nucleotide was biphasic. This was reflected in an increased K{dollar}\sb{lcub}\rm m{rcub}{dollar} for de novo incorporation of one nucleotide at a single nucleotide gap (0.7mM), as opposed to the K{dollar}\sb{lcub}\rm m{rcub}{dollar} for incorporation of one nucleotide into singly primed M13 DNA (0.18mM).



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