Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).     

Characterization of the DNA polymerases induced by a group of herpes simplex virus type I variants selected for growth in the presence of phosphonoformic acid

Five independently derived variants of a herpes simplex virus type I (HSV-1) strain were plaque purified from a virus population passaged in 1 mM phosphonoformic acid (PFA). The DNA polymerase induced by the parent and PFA-resistant viruses were purified and characterized. No differences were observed among the enzymes with respect to their chromatographic properties, specific activities, or polypeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The variant enzymes exhibited levels of PFA resistance which ranged from 15- to 25-fold. Resistance to PFA was always associated with a similar degree of resistance to its congener phosphonoacetic acid, but cross-resistance to beta-phenylphosphonoacetic acid was only seen with two of the five variant enzymes. PFA and pyrophosphate were mutually competitive in PPi exchange reactions, but in DNA synthetic reactions the levels of resistance to PFA and PPi were not equal. The apparent affinities of the enzymes for Mg2+ did parallel their affinities for PFA. Km values of dNTPs were about 2-fold higher than the parent virus enzyme for all of the variant enzymes except one which was 4-fold higher. The processivity of polymerization was apparently unaffected by the enzyme changes related to PFA resistance although one variant enzyme had a lower value. Resistance among the variant enzymes to the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 2',3'-dideoxyguanosine was directly related to the level of resistance to PFA. The data presented here indicated that (i) PFA resistance may result from several types of active site alterations, since the PFA-resistant enzymes were of three kinetically distinct types. Also, additional enzyme alterations, probably unrelated to PFA resistance, were detected in one enzyme. (ii) PFA and PPi possess some different binding determinants within the active center of herpes simplex virus type I DNA polymerase. (iii) PFA and the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 2',3'-dideoxyguanosine may have a common ultimate inhibitory mechanism.