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

Chen SH, Suzuki CK, Wu SH - Nucleic Acids Res. (2008)

Bottom Line: Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

ABSTRACT
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.

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Formation of G-quartets in LSPas and TG6T demonstrated by circular dichroism spectroscopy. CD spectra of LSPas (A and B) and TG6T (C and D) were monitored from 10°C to 90°C in the presence or absence of urea. The buffer used in A and C contains 10 mM sodium cacodylate (pH 7.5), 100 mM NaCl, 5 mM MgCl2 and 0.1 mM EDTA. The buffer used in B and D consists of 10 mM sodium cacodylate (pH 7.5) and 1 M urea.
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Figure 2: Formation of G-quartets in LSPas and TG6T demonstrated by circular dichroism spectroscopy. CD spectra of LSPas (A and B) and TG6T (C and D) were monitored from 10°C to 90°C in the presence or absence of urea. The buffer used in A and C contains 10 mM sodium cacodylate (pH 7.5), 100 mM NaCl, 5 mM MgCl2 and 0.1 mM EDTA. The buffer used in B and D consists of 10 mM sodium cacodylate (pH 7.5) and 1 M urea.

Mentions: Recent work suggests that human Lon binds to mtDNA in living cells and preferentially associates with the control region (CR) of the mitochondrial genome (28). The putative mtDNA-binding target of hLon within CR overlaps the light strand promoter (LSP) and corresponds to the previously characterized oligonucleotide sequence referred to as LSPas. LSPas is a 24-base sequence located on the heavy (or antisense) strand of mtDNA (5′-AATAATGTGTTAGTTGGGGGGTGA-3′). Notably, the mtDNA heavy strand has a higher content of guanine and thymine residues, in contrast to the complementary light strand (44). LSPas contains six contiguous guanine bases, rendering a high-order structure demonstrated by electrophoretic analysis using both native and denaturing gels (Figure 1). Two forms of LSPas were observed (Figure 1A, lane 2): a monomeric form that migrated slightly slower than the complementary LSPs CA-containing oligonucleotide (lane 1) and a high-order structure that migrated slower than the double-stranded LSP (compare lanes 2 and 3). Similar migration differences were also observed for an 18-mer counterpart (LSPas18) that lacks six bases from the 5′ end of LSPas but retains the G-rich core sequence. The slower migrating forms of both LSPas and LSPas18 remained unchanged on denaturing polyacrylamide gels containing urea (Figure 1B, lanes 2 and 5, respectively), suggesting the presence of a structural motif, which was later identified by CD (Figure 2). Similarly, an 8-mer TG6T migrated much slower than its complementary AC6A (Figure S1, lanes 5 and 7, respectively). TG6T was unaffected by heat treatment at 120°C for 2 h, whereas the slower migrating form of LSPas was disrupted after heat treatment (Figure S1, compare lane 5 with 6, and lane 1 with 2). The susceptibility to heat denaturation may be attributed to the length of the ssDNA oligonucleotide, in addition to the number of bases flanking the G-rich core sequence.Figure 1.


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

Chen SH, Suzuki CK, Wu SH - Nucleic Acids Res. (2008)

Formation of G-quartets in LSPas and TG6T demonstrated by circular dichroism spectroscopy. CD spectra of LSPas (A and B) and TG6T (C and D) were monitored from 10°C to 90°C in the presence or absence of urea. The buffer used in A and C contains 10 mM sodium cacodylate (pH 7.5), 100 mM NaCl, 5 mM MgCl2 and 0.1 mM EDTA. The buffer used in B and D consists of 10 mM sodium cacodylate (pH 7.5) and 1 M urea.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275097&req=5

Figure 2: Formation of G-quartets in LSPas and TG6T demonstrated by circular dichroism spectroscopy. CD spectra of LSPas (A and B) and TG6T (C and D) were monitored from 10°C to 90°C in the presence or absence of urea. The buffer used in A and C contains 10 mM sodium cacodylate (pH 7.5), 100 mM NaCl, 5 mM MgCl2 and 0.1 mM EDTA. The buffer used in B and D consists of 10 mM sodium cacodylate (pH 7.5) and 1 M urea.
Mentions: Recent work suggests that human Lon binds to mtDNA in living cells and preferentially associates with the control region (CR) of the mitochondrial genome (28). The putative mtDNA-binding target of hLon within CR overlaps the light strand promoter (LSP) and corresponds to the previously characterized oligonucleotide sequence referred to as LSPas. LSPas is a 24-base sequence located on the heavy (or antisense) strand of mtDNA (5′-AATAATGTGTTAGTTGGGGGGTGA-3′). Notably, the mtDNA heavy strand has a higher content of guanine and thymine residues, in contrast to the complementary light strand (44). LSPas contains six contiguous guanine bases, rendering a high-order structure demonstrated by electrophoretic analysis using both native and denaturing gels (Figure 1). Two forms of LSPas were observed (Figure 1A, lane 2): a monomeric form that migrated slightly slower than the complementary LSPs CA-containing oligonucleotide (lane 1) and a high-order structure that migrated slower than the double-stranded LSP (compare lanes 2 and 3). Similar migration differences were also observed for an 18-mer counterpart (LSPas18) that lacks six bases from the 5′ end of LSPas but retains the G-rich core sequence. The slower migrating forms of both LSPas and LSPas18 remained unchanged on denaturing polyacrylamide gels containing urea (Figure 1B, lanes 2 and 5, respectively), suggesting the presence of a structural motif, which was later identified by CD (Figure 2). Similarly, an 8-mer TG6T migrated much slower than its complementary AC6A (Figure S1, lanes 5 and 7, respectively). TG6T was unaffected by heat treatment at 120°C for 2 h, whereas the slower migrating form of LSPas was disrupted after heat treatment (Figure S1, compare lane 5 with 6, and lane 1 with 2). The susceptibility to heat denaturation may be attributed to the length of the ssDNA oligonucleotide, in addition to the number of bases flanking the G-rich core sequence.Figure 1.

Bottom Line: Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus