Thermodynamic characterization of specific interactions between the human Lon protease and G-quartet DNA

Lon is an ATP-powered protease that binds DNA. However, the function of DNA binding by Lon remains elusive. Studies suggest that human Lon (hLon) binds preferentially to a G-rich single-stranded DNA (ssDNA) sequence overlapping the light strand promoter of mitochondrial DNA. This sequence is contained within a 24-base oligonucleotide referred to as LSPas. Here, we use biochemical and biophysical approaches to elucidate the structural properties of ssDNAs bound by hLon, as well as the thermodynamics of DNA binding by hLon. Electrophoretic mobility shift assay and circular dichroism show that ssDNAs with a propensity for forming parallel G-quartets are specifically bound by hLon. Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction in heat capacity. Differential scanning calorimetry pinpoints an excess heat capacity upon hLon binding to LSPas. By contrast, hLon binding to an 8-base G-rich core sequence is entropically driven with a relatively negligible change in heat capacity. A considerable enhancement of thermal stability accompanies hLon binding to LSPas as compared to the G-rich core. Taken together, these data support the notion that hLon binds G-quartets through rigid-body binding and that binding to LSPas is coupled with structural adaptation.     

Fission yeast Dna2 is required for generation of the telomeric single-strand overhang

It has been suggested that the Schizosaccharomyces pombe Rad50 (Rad50-Rad32-Nbs1) complex is required for the resection of the C-rich strand at telomere ends in taz1-d cells. However, the nuclease-deficient Rad32-D25A mutant can still resect the C-rich strand, suggesting the existence of a nuclease that resects the C-rich strand. Here, we demonstrate that a taz1-d dna2-2C double mutant lost the G-rich overhang at a semipermissive temperature. The amount of G-rich overhang in S phase in the dna2-C2 mutant was lower than that in wild-type cells at the semipermissive temperature. Dna2 bound to telomere DNA in a chromatin immunoprecipitation assay. Moreover, telomere length decreased with each generation after shift of the dna2-2C mutant to the semipermissive temperature. These results suggest that Dna2 is involved in the generation of G-rich overhangs in both wild-type cells and taz1-d cells. The dna2-C2 mutant was not gamma ray sensitive at the semipermissive temperature, suggesting that the ability to process double-strand break (DSB) ends was not affected in the dna2-C2 mutant. Our results reveal that DSB ends and telomere ends are processed by different mechanisms.