Electron Microscopic Analysis of the Products of DNA Synthesis by DNA Polymerases from Calf Thymus and Herpes Simplex Virus Type I.

摘要

Replication of single stranded M 13 DNA by the calf thymus DNApolymerase alpha:primase complex and herpes simplex virus type 1(HSV-1) DNA polymerase was examined by electron microscopy. The replicative intermediates isolated from the reaction were spread for electron microscopy in the presence of E. Coli single stranded binding (SSB) protein and ethidium bromide. The electron microscopic analysis of the replicative intermediates from the calf thymus DNA primase primed M13 DNA replication showed an average of 2.5 primers per M13 DNA circle. The measurement of the double stranded length from individual replicative intermediates by electron microscopy was within the accuracy of 10% standard deviation. The product length distribution obtained from the HSV-1 DNA polymerase catalyzed replication of M13 DNA primed with a specific pentadecamer and in the presence of E. Coli SSB protein showed a near Poisson distribution. Replication of the same primer-template system or DNA primase primed M13 DNA template by calf thymus DNA polymerase a showed a broad distribution of product lengths. Analysis of replicative intermediates after digestion with restriction endonuclease Hae Ill showed that replication of singly primed M13 by calf thymus DNA polymerase a is affected by a major hairpin structure located at the origin of replication. Elongation of a primer at an aberrant priming site located upstream from the primary priming site was observed during replication. HSV-1 DNA polymerase replication in the presence of E. Coli SSB appeared not to be affected by this hairpin structure as analyzed by Hae Ill Restriction digestion. Electron microscopic analysis of early replication products synthesized by HSV-1 DNA polymerase and calf thymus DNA polymerase a:primase complex showed that the majority of products were partially replicated. Product analysis of reactions with DNA template concentrations increased 2-3 fold also showed partially replicated structures with no full length products.