Rajesh Kasiviswanathan

Regulation of Minichromosome Maintenance Helicase Activity by Cdc6

Genetic studies, together with amino acid and structural similarities to the clamp loaders of DNA polymerase sliding clamps, have suggested that the Cdc6 protein may function as a loader for the eukaryotic replicative helicase, the minichromosome maintenance (MCM) complex. Thus, Cdc6 may act as the functional homologue of the bacterial DnaC that utilizes ATP hydrolysis to assemble the DnaB helicase at the origin. This report shows that the helicase activity of an MCM homologue from the archaeon Methanothermobacter thermautotrophicus is inhibited in the presence of the Cdc6 homologues. This inhibitory activity is dependent, as for DnaC, on ATP binding to Cdc6. Moreover, an intact Cdc6 winged helix domain is required for efficient inhibition. Two-hybrid analyses indicated that MCM and Cdc6 interact and that the interaction is mediated by the winged helix domain. Analysis of Cdc6 and MCM homologues from several archaea exhibited differences in the inhibitory activity suggesting divergence in function in Cdc6 and MCM homologues among the archaea.

RECQL4 localizes to mitochondria and preserves mitochondrial DNA integrity

RECQL4 is associated with Rothmund–Thomson Syndrome (RTS), a rare autosomal recessive disorder characterized by premature aging, genomic instability, and cancer predisposition. RECQL4 is a member of the RecQ helicase family, and has many similarities to WRN protein, which is also implicated in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present in mitochondria. Q‐PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4‐deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial reserve capacity after lentiviral knockdown of RECQL4 in two different primary cell lines. Additionally, biochemical assays with RECQL4, mitochondrial transcription factor A, and mitochondrial DNA polymerase γ showed that the polymerase inhibited RECQL4’s helicase activity. RECQL4 is the first 3′–5′ RecQ helicase to be found in both human and mouse mitochondria, and the loss of RECQL4 alters mitochondrial integrity.

Biochemical analysis of human POLG2 variants associated with mitochondrial disease

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic.

Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2′-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3–14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3′-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed.

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

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol γ), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol γ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g→t and T885S, c.2653a→t). Biochemical characterization of purified, recombinant forms of pol γ 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% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol γ 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 γ and examines the consequences of mitochondrial disease mutations in this region.

The interface of transcription and DNA replication in the mitochondria

DNA replication of the mitochondrial genome is unique in that replication is not primed by RNA derived from dedicated primases, but instead by extension of processed RNA transcripts laid down by the mitochondrial RNA polymerase. Thus, the RNA polymerase serves not only to generate the transcripts but also the primers needed for mitochondrial DNA replication. The interface between this transcription and DNA replication is not well understood but must be highly regulated and coordinated to carry out both mitochondrial DNA replication and transcription. This review focuses on the extension of RNA primers for DNA replication by the replication machinery and summarizes the current models of DNA replication in mitochondria as well as the proteins involved in mitochondrial DNA replication, namely, the DNA polymerase γ and its accessory subunit, the mitochondrial DNA helicase, the single-stranded DNA binding protein, topoisomerase I and IIIα and RNaseH1. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

R964C Mutation of DNA Polymerase γ Imparts Increased Stavudine Toxicity by Decreasing Nucleoside Analog Discrimination and Impairing Polymerase Activity

ABSTRACT The R964C mutation of human DNA polymerase γ was recently linked to stavudine (d4T)-mediated mitochondrial toxicity. We utilized pre-steady-state kinetics to determine the effect of this mutation on incorporation of natural substrate dTTP and the active metabolite of d4T (d4TTP). The R964C polymerase γ holoenzyme demonstrated a 33% decrease in dTTP incorporation efficiency and a threefold-lower d4TTP discrimination relative to that of the wild-type polymerase γ, providing a mechanistic basis for genetic predisposition to nucleoside reverse transcriptase inhibitor toxicity.

Human Mitochondrial DNA Polymerase γ Exhibits Potential for Bypass and Mutagenesis at UV-induced Cyclobutane Thymine Dimers

Background: Thymine-thymine dimers (T-T) are an important form of ultraviolet radiation-induced DNA damage. Results: Human mitochondrial DNA polymerase γ demonstrated a low level of translesion synthesis past T-T that was further attenuated by exonuclease activity but was frequently mutagenic. Conclusion: Such damage may inhibit mitochondrial DNA replication and contribute to mutagenesis in vivo. Significance: Alterations in mitochondrial DNA maintenance are associated with skin cancer. Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T. We performed in vitro DNA polymerization assays to measure the kinetics of incorporation opposite the lesion and bypass of the lesion by pol γ with a dimer-containing template. Exonuclease-deficient pol γ bypassed thymine dimers with low relative efficiency; bypass was attenuated but still detectable when using exonuclease-proficient pol γ. When bypass did occur, pol γ misincorporated a guanine residue opposite the 3′-thymine of the dimer only 4-fold less efficiently than it incorporated an adenine. Surprisingly, the pol γ exonuclease-proficient enzyme excised the incorrectly incorporated guanine at similar rates irrespective of the nature of the thymines in the template. In the presence of all four dNTPs, pol γ extended the primer after incorporation of two adenines opposite the lesion with relatively higher efficiency compared with extension past either an adenine or a guanine incorporated opposite the 3′-thymine of the T-T. Our results suggest that T-T usually stalls mitochondrial DNA replication but also suggest a mechanism for the introduction of point mutations and deletions in the mitochondrial genomes of chronically UV-exposed cells.

Biochemical analysis of the G517V POLG variant reveals wild-type like activity

The c.1550g→t mutation in the POLG gene causing the G517V substitution has been reported by many groups to be associated with a variety of mitochondrial diseases, including autosomal dominant and recessive forms of ataxia neuropathy, myopathy and microcephaly, progressive external ophthalmoplegia, diabetes, strokes, hypotonia, and epilepsy. However, the variable disease presentation and age of onset raises suspicion of its pathogenicity. Because of the varied reported associated symptoms and request from physicians to address the consequence of this mutation, we have carried out the biochemical analysis of the purified recombinant human DNA polymerase γ protein harboring the G517V substitution. These analyses revealed that the G517V mutant enzyme retained 80-90% of wild-type DNA polymerase activity, in addition to its functional interaction with the p55 accessory subunit. DNA binding by the mutant was also only slightly lower than the wild-type enzyme. Our data suggest that the G517V mutation by itself in pol γ most likely does not have a role in mitochondrial disorders.

Mechanism of Interaction of Human Mitochondrial DNA Polymerase γ with the Novel Nucleoside Reverse Transcriptase Inhibitor 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine Indicates a Low Potential for Host Toxicity

ABSTRACT The potent antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a promising experimental agent for treating HIV infection. Pre-steady-state kinetics were used to characterize the interaction of EFdA-triphosphate (EFdA-TP) with human mitochondrial DNA polymerase γ (Pol γ) to assess the potential for toxicity. Pol γ incorporated EFdA-TP 4,300-fold less efficiently than dATP, with an excision rate similar to ddATP. This strongly indicates EFdA is a poor Pol γ substrate, suggesting minimal Pol γ-mediated toxicity, although this should be examined under clinical settings.