Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

Abstract

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol ␥), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol ␥ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g3a; T851A, c.2551a3g; R852C, c.2554c3t; R853Q, c.2558g3a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g3t and T885S, c.2653a3t). Biochemical characterization of purified, recombinant forms of pol ␥ revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50 -70% wildtype polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol ␥ accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol ␥ and examines the consequences of mitochondrial disease mutations in this region.As the only DNA polymerase found in animal cell mitochondria, DNA polymerase ␥ (pol ␥) 3 bears sole responsibility for DNA synthesis in all replication and repair transactions involving mitochondrial DNA (1, 2). Mammalian cell pol ␥ is a heterotrimeric complex composed of one catalytic subunit of 140 kDa (p140) and two 55-kDa accessory subunits (p55) that form a dimer (3). The catalytic subunit contains an N-terminal exonuclease domain connected by a linker region to a C-terminal polymerase domain. Whereas the exonuclease domain contains essential motifs I, II, and III for its activity, the polymerase domain comprising the thumb, palm, and finger subdomains contains motifs A, B, and C that are crucial for polymerase activity. The catalytic subunit is a family A DNA polymerase that includes bacterial pol I and T7 DNA polymerase and possesses DNA polymerase, 3Ј 3 5Ј exonuclease, and 5Ј-deoxyribose phosphate lyase activities (for review, see Refs. 1 and 2). The 55-kDa accessory subunit (p55) confers processive DNA synthesis and tight binding of the pol ␥ complex to DNA (4, 5).Depletion of mtDNA as well as the accumulation of deletions and point mutations in mtDNA have been observed in several mitochondrial disorders (for review, see Ref. 6). mtDNA depletion syndromes are caused by defects in nuclear genes responsible for replication and maintenance of the mitochondrial genome (7). Mutation of POLG, the gene encoding the catalytic subunit of pol ␥, is frequently involved in disorders linked to mutagenesis of mtDNA (8, 9). Presently, more than 150 point mutations in POLG are linked with a wide variety of mitochondrial diseases, including the autosomal dominant (ad) and...

Defects of Mitochondrial DNA Replication

“…23 Analysis of a cluster of Alpers mutations in the thumb domain has shown a striking correlation with the severity of the defect and the degree of conservation of amino acid sequences among various eukaryotes. 48 Mutations in the most conserved sites represented by G848S, T851A, R852C, and R853Q exhibited less than 1% wild-type enzyme activity. 48 Mutations in codons for less conserved amino acids (Gln879 and Thr885) resulted in only moderate reduction in activity.…”

Yeast mitochondrial DNA polymerase is a highly processive single-subunit enzyme

“…As the only DNA polymerase found in mitochondria, Polγ is solely responsible for all DNA synthesis in the organelle during replication as well as repair (Graziewicz et al, 2006). Thus, malfunction of Polγ leads to severe mitochondrial disorders in human (Van Goethem et al, 2001;Graziewicz et al, 2004;Longley et al, 2005;Kasiviswanathan et al, 2009).…”

Biophysical studies of mutated K562 DNA (erythroleukemic cells) binding to adriamycin and daunomycin reveal that mutations induce structural changes influencing binding behavior

“…Mutations leading to structural changes influence binding of Pilin antigen have been observed in CD spectra for synthetic DNA, rich in guanine moiety (Cahoon & Seifert, 2009). Disruption in structure of proteins on point mutations has been widely studied by CD and reported, as it is highly relevant for protein DNA binding (Kasiviswanathan, Longley, Chan, & Copeland, 2009;Ohmae, Sasaki, & Gekko, 2001). The negative peak at 240-250 nm is characteristic feature in CD spectra of B form DNA.…”