Stimulation of Drosophila Mitochondrial DNA Polymerase by Single-stranded DNA-binding Protein

Abstract

Mitochondrial DNA polymerase from Drosophila embryos has been characterized with regard to its mechanism of DNA synthesis in the presence of single-stranded DNA-binding protein from Escherichia coli. The rate of DNA synthesis by DNA polymerase gamma was increased nearly 40-fold upon addition of single-stranded DNA-binding protein. Processivity of mitochondrial DNA polymerase was increased approximately 2-fold, while its intrinsic rate of nucleotide polymerization was unaffected. Primer extension analysis showed that the rate of initiation of DNA strand synthesis by DNA polymerase gamma was increased 25-fold in the presence of single-stranded DNA-binding protein. Our results indicate that the stimulation of Drosophila DNA polymerase gamma by single-stranded DNA-binding protein results primarily from an increased rate of primer recognition and binding. Concurrent achievement of maximal activity and processivity by mitochondrial DNA polymerase in the presence of binding protein suggests that DNA polymerase gamma, like other replicative DNA polymerases, associates with accessory factors in vivo to catalyze efficient and processive DNA synthesis.

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

“…In parallel with the stimulation profiles, we observed that mtSSB predominantly enhances primer utilization to increase the extent of DNA synthesis, with only a modest effect on DNA product strand length, which we determined in this analysis to be ϳ760 nt in the absence of mtSSB up to ϳ1100 nt in its presence. This effect is similar to that described previously for Pol ␥ from D. melanogaster and with the processivity of human Pol ␥ calculated from single nucleotide incorporation assays (12,22,23).…”

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

“…We would suggest that this derives from a different mechanism, because both these and the parent deletion mutant ⌬␥3 retain strong subunit interactions that are comparable with wild-type pol ␥. One interesting possibility is that the reduced processivity of the L558A and F578A mutants is related to defective interaction with mtSSB, a factor that increases strongly both the activity and processivity of wild-type DNA pol ␥ (37,39). Notably, the stimulation of DNA polymerase activity on M13 DNA by mtSSB is reduced 4 -5-fold in the L558A and F578A mutant holoenzymes as compared with wild type (6 -7-versus 30-fold stimulation).…”

Physiological and Biochemical Defects in Functional Interactions of Mitochondrial DNA Polymerase and DNA-binding Mutants of Single-stranded DNA-binding Protein

“…The stimulatory effects of mtSSB are minimal at higher salt concentrations where activity is high and processivity is reduced. Although wild-type mtSSB enhances proces- 4 , dsRNA and exogenous mtSSB mRNA were induced. Induction of dsRNA triggers degradation of endogenous mtSSB mRNA.…”

“…7 was determined by Southern blot analysis. DNAs were isolated as described under "Experimental Procedures" after growth for 10 days in the presence or absence of 0.4 mM CuSO 4 . A fragment of the ATPase6 gene was used as the probe for determination of mtDNA content, and a DNA fragment from the nuclear histone gene cluster was used for normalization.…”

“…The standard assay for stimulation of pol ␥ by mtSSB on single-stranded DNA substrates is performed at 30 mM KCl, where maximal DNA synthesis combined with the highest enzyme processivity is observed (4,5). Under this ionic condition, all of the mtSSB variants with single amino acid substitutions displayed DNA-binding activities Ն 90% of wild type, whereas the double mutant W79T/F85A showed 50% of wild-type DNA binding (Fig.…”