ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia

De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.Peer reviewe

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Funder: Queensland GovernmentSyphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides

Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol

Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37 degrees C. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol

Isoforms of Base Excision Repair Enzymes Produced by Alternative Splicing

Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport

Purification and the catalytic activity of PrimPol from different expression systems.

<p><b>(</b>A) purified preparation of GST-tagged PrimPol from different <i>E</i>. <i>coli</i> strains and <i>S</i>. <i>cerevisiae</i>. Coomassie staining. (B) the comparison of the DNA polymerase activity of PrimPol purified from different strains of <i>E</i>. <i>coli</i>. 75–450 nM of GST-tagged PrimPol and 100 nM 30-mer DNA template were used in the reactions. (C) purified preparation of PrimPol from human FreeStyle 293-F cells. Coomassie staining. (D) the comparison of the DNA polymerase activity of PrimPol purified from ArcticExpress(DE3)-pRIL <i>E</i>. <i>coli</i> cells and human FreeStyle 293-F cells. 200 nM of PrimPol and 25 nM 70-mer DNA template were used in the reactions. Experiments (B and D) were repeated four times.</p

The reaction buffer composition affects the DNA polymerase activity of PrimPol <i>in vitro</i>.

<p>(A) the DNA polymerase activity of PrimPol at different NaCl concentrations. (B) the DNA polymerase activity of PrimPol at different pH. HEPES based buffer was used to test the DNA polymerase activity of PrimPol at different pH values. (C) the DNA polymerase activity of PrimPol at different MgCl₂ and MnCl₂ concentrations. 200 nM of PrimPol and 25 nM 70-mer DNA template were used in the reactions. All experiments were repeated three times.</p

Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol - Table 1

<p>Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol</p> - Table

The stability of the PrimPol’s catalytic activity <i>in vitro</i>.

<p>(A) the DNA polymerase activity of PrimPol after the indicated time pre-incubation on ice. (B) the DNA polymerase activity of PrimPol after the indicated amount of freeze-thaw cycles. (C) the DNA polymerase activity of PrimPol after 30 min incubation at 0ºC, 25ºC, 30ºC, 35ºC, 37ºC and 40ºC. (D) the DNA polymerase activity of PrimPol after incubation at 37ºC. 450 nM of PrimPol and 100 nM 30-mer DNA template were used in the reactions. Experiments were repeated two times in A and B and three times in C and D.</p