Human Mitochondrial DNA Polymerase Metal Dependent UV Lesion Bypassing Ability

Abstract

Human mitochondrial DNA contains more UV-induced lesions than the nuclear DNA due to lack of mechanism to remove bulky photoproducts. Human DNA polymerase gamma (Pol γ) is the sole DNA replicase in mitochondria, which contains a polymerase (pol) and an exonuclease (exo) active site. Previous studies showed that Pol γ only displays UV lesion bypassing when its exonuclease activity is obliterated. To investigate the reaction environment on Pol γ translesion activity, we tested Pol γ DNA activity in the presence of different metal ions. While Pol γ is unable to replicate through UV lesions on DNA templates in the presence of Mg²⁺, it exhibits robust translesion DNA synthesis (TLS) on cyclobutane pyrimidine dimer (CPD)-containing template when Mg²⁺ was mixed with or completely replaced by Mn²⁺. Under these conditions, the efficiency of Pol γ's TLS opposite CPD is near to that on a non-damaged template and is 800-fold higher than that of exonuclease-deficient Pol γ. Interestingly, Pol γ exhibits higher exonuclease activity in the presence of Mn²⁺ than with Mg²⁺, suggesting Mn²⁺-stimulated Pol γ TLS is not via suppressing its exonuclease activity. We suggest that Mn²⁺ ion expands Pol γ's pol active site relative to Mg²⁺ so that a UV lesion can be accommodated and blocks the communication between pol and exo active sites to execute translesion DNA synthesis.

Keywords: DNA polymerase gamma; TLS; UV lesion; metal-dependence; mitochondrial DNA.

FIGURE 1

DNA synthesis of wild-type Pol γ on non-damaged and CPD-containing templates in the presence of Mg²⁺ or Mn²⁺. (A) Structure of thymine bases, CPD, and (6-4)PP where nitrogen is represented in blue spheres, oxygen in red spheres, carbon in black spheres, and phosphorus in golden spheres. (B) Time-dependent DNA synthesis of wild-type Pol γ on 5′-³²P-Primer N (P_N) annealed to a non-damaged template (T-ND) (lanes 1–8) or CPD-containing template (T-CPD) (lanes 9–16) in the presence of Mg²⁺. (C) Time-dependent DNA synthesis of wild-type Pol γ on 5′-³²P-P_N annealed to T-ND (lanes 1–8) or T-CPD (lanes 9–16) in the presence of Mn²⁺. (D) Quantification of normalized full-length (FL) products from (B) and (C) over time in seconds. The results are presented as mean and standard deviation from three independent experiments.

FIGURE 2

DNA synthesis of exonuclease-deficient Pol γ on non-damaged and CPD-containing templates in the presence of Mg²⁺ or Mn²⁺. (A) Time-dependent DNA synthesis of exonuclease-deficient (exo-) Pol γ on 5′-³²P-P_N annealed to T-ND in the presence of Mg²⁺ (lanes 1–8) or Mn²⁺ (lanes 9–16) (B) Time-dependent DNA synthesis of Pol γ exo-on 5′-³²P-P_N annealed to T-CPD in the presence of Mg²⁺ (lanes 1–8) or Mn²⁺ (lanes 9–16). (C) Quantification of normalized full-length (FL) products from (A) and (B) over time in seconds. The results are presented as mean and standard deviation from three independent experiments.

FIGURE 3

Metal-dependent DNA synthesis of wild-type and exonuclease-deficient Pol γ on a (6-4)PP-containing template. Time-dependent polymerization assay of wild-type Pol γ (exo+) on 5′-³²P-P_N annealed to T-(6–4)PP in the presence of Mg²⁺ (lanes 2 and 3) or in the presence of Mn²⁺ (lanes 4 and 5) and of Pol γ (exo-) on identical DNA substrates in the presence of Mg²⁺ (lanes 7 and 8) or Mn²⁺ (lanes 9 and 10).

FIGURE 4

Metal-regulated exonuclease activity of wild-type Pol γ. (A) Exonuclease assay of Pol γ on a single-stranded DNA, 5′-³²P-P_N, in the presence of Mg²⁺ (lanes 1–8) or in the presence of Mn²⁺ (lanes 9–16). (B) Exonuclease assay Pol γ on primer/template, 5′-³²P-P_N/T-ND, in the presence of Mg²⁺ (lanes 1–8) or in the presence of Mn²⁺ (lanes 9–16) (C). Quantification of normalized fraction of substrate remaining in (A) and (B) over time in seconds shown. The results are presented as mean and standard deviation from three independent experiments. Shorter time points (0–200 s) are shown as an inset.

FIGURE 5

Effect of a single metal ion on wild-type Pol γ DNA activity on non-damaged and CPD-containing templates. (A) Wild-type Pol γ primer extension on T-ND annealed to 5′-³²P-PN in the presence of Mg²⁺(lanes 1–9) or Mn²⁺ (lanes 10-18). (B) Quantification of the full-length product on T-ND vs. a gradient of Mg²⁺ or Mn²⁺ shown in (A). (C) Wild-type Pol γ primer extension on T-CPD annealed to 5′-³²P-PN in the presence of Mg²⁺ (lanes 1–9) or Mn²⁺ (lanes 10-18). (D) Quantification of the full-length product on T-CPD vs. a gradient of Mg²⁺ or Mn²⁺ shown in (C).

FIGURE 6

Effect of mixed metal ions on wild-type Pol γ DNA synthesis on non-damaged and CPD-containing templates in a mixture of Mg²⁺ and Mn²⁺. (A) Wild-type Pol γ primer extension on T-ND (lanes 1–9) or T-CPD (lanes 10–18) annealed to the 5′-³²P-P_N at a constant concentration of Mg²⁺ and varied concentrations of Mn²⁺. (B) Quantification of the full-length product vs. concentration of Mn²⁺ shown in (A). (C) Wild-type Pol γ primer extension on T-ND (lanes 1–9) or T-CPD (lanes 10–18) annealed to the 5′-³²P-P_N at constant concentration of Mn²⁺ and varied concentrations of Mg²⁺. (D) Quantification of the full-length product vs. concentration of Mg²⁺ shown in (C).

FIGURE 7

Evaluation of wild-type T7 polymerase activity on a non-damaged and CPD substrates in the presence of Mg²⁺ or Mn²⁺. (A) Time course activity assay of wild-type T7 DNA polymerase on T-ND (lanes 1–8) and on T-CPD (lanes 9–16) annealed to 5′- ³²P-P_N in the presence of Mg²⁺. (B) Time course activity assay of wild-type T7 DNA polymerase on T-ND (lanes 1–8) and on T-CPD (lanes 9–16) annealed to 5′-³²P-P_N in the presence of Mn²⁺.

FIGURE 8

Comparison of pol site conformation of Pol η and Pol γ. Left, Pol η ternary complex with CPD (red)-containing template (blue) and primer (magenta) and incoming nucleotide (green) (PDB: 3MR3). Middle, modeled Pol γ-CPD containing primer/template DNA showing the CPD (red) steric clashes with the O helices. Right, Pol γ ternary complex with primer/template and incoming nucleotide showing the bending of the template by O- and O1-helix.

FIGURE 9

Schematic model of the hypothetical function of Mg²⁺ and Mn²⁺ in reshaping Pol γ′s active site. Illustration of Pol γ (green) on an ND- (blue trapezoid) or CPD-containing template (violet rhomboid). (A) Representation of DNA replication by Pol γ on an ND-containing substrate in the presence of Mg²⁺, Mg²⁺/Mn²⁺, or Mn²⁺ showing full extension in all cases without significant changes in the catalytic site (yellow trapezoid). (B) Representation of DNA replication by Pol γ on a CPD-containing template illustrating a change of the Pol γ′s catalytic site (yellow trapezoid) in the presence of Mn²⁺ upon encountering CPD to trigger TLS activity.