Human Mitochondrial DNA Polymerase Metal Dependent UV Lesion Bypassing Ability.

Joon Park,Noe Baruch-Torres,Luis G Brieba,Shigenori Iwai,Geoffrey K Herrmann,Y Whitney Yin

doi:10.3389/fmolb.2022.808036

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.

Highlights

Ultraviolet (UV) exposure of DNA causes dimerization of two adjacent pyrimidines, forming cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidones ((6-4)PP), which stalls replicating polymerases (Varghese and Wang, 1967a; Varghese and Wang, 1967b; Brash and Haseltine, 1982)
Human mitochondria DNA is replicated by DNA polymerase gamma (Pol γ), which consists of a catalytic subunit Pol γA and a dimeric accessory subunit Pol γB
DNA templates (49 nt) containing either a CPD (T-CPD) or (6-4)PP (T-(6-4)PP) (Table 1) at the 28th position from the 3′end was synthesized as described in Methods

Summary

Introduction

Ultraviolet (UV) exposure of DNA causes dimerization of two adjacent pyrimidines, forming cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidones ((6-4)PP), which stalls replicating polymerases (Varghese and Wang, 1967a; Varghese and Wang, 1967b; Brash and Haseltine, 1982). Human mitochondria lack nucleotide excision repair or photolyase that removes the photoproducts (Maher et al, 1976; Maher et al, 1979; Maher et al, 1982); mitochondrial DNA replication machinery would inevitably encounter the UV photoproducts. Human mitochondria DNA is replicated by DNA polymerase gamma (Pol γ), which consists of a catalytic subunit Pol γA and a dimeric accessory subunit Pol γB. Pol γ possesses activities of 5′-3′ polymerization (pol) for DNA synthesis, 3′-5′ exonuclease (exo) for proofreading, and 5′deoxyribose phosphate lyase for DNA repair. Pol γB has no intrinsic enzymatic activity, but it accelerates polymerization rate, increases affinity to DNA, and enhances processivity of the holoenzyme (Johnson et al, 2000; Johnson and Johnson, 2001; Lee et al, 2010).

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Conclusion