TOP3A Mutations Lead to Markedly Reduced Cellular Amounts of Enzyme TopIIIα
Most of the identified TOP3A mutations are predicted to prematurely truncate the encoded protein (Figure 2A) and hence are likely to have significant effects on cellular protein amounts. We therefore assessed the amounts of TopIIIα in primary dermal fibroblast cell lines derived from P1, P7, and P8. Immunoblotting demonstrated that the amount of full-length TopIIIα was substantially lower in total cell lysates from all three individuals than in cell lines from unrelated control individuals and parents (Figure 2B).
Figure 2 Mutations in TOP3A Markedly Reduce Amounts of TopIIIα but Do Not Affect Its Intrinsic Decatenation Activity
(A) Schematic of TopIIIα with locations of variants annotated. Colors are as follows: red, TOPRIM domain; purple, TOPA domain; and blue, GRF zinc-finger domain.
(B) TOP3A mutations markedly reduce protein amounts. An immunoblot of a polyacrylamide gel is shown and was generated from total cell lysates from dermal fibroblast lines derived from affected individuals probed with an anti-TopIIIα antibody. siRNA depletion of TOP3A in a fibroblast line from control 2 was used to confirm the specificity of the antibody. C1 and C2 are unrelated control fibroblast lines. TopIIIα antibody was raised against amino acids 652–1,001.
(C) Recombinant TopIIIαThr812LeufsTer101 (denoted TopIIIαP1) is less stable than TopIIIαWT. An immunoblot of a polyacrylamide gel is shown and was generated from purified TopIIIαWT and TopIIIαP1 proteins probed with an anti-TopIIIα antibody. Protein size markers are 250, 170, 140, 100, and 70 kDa.
(D) Schematic depicting the BTRR complex and its role in dHJ dissolution. Homologous sister chromatids are shown in red and blue.
(E and F) TopIIIαP1 is proficient for promoting dHJ dissolution with BLM in combination with RMI1 and RMI2. (E) Representative polyacrylamide gel demonstrating the enzyme-concentration dependence of the dHJ dissolution activity of TopIIIαWT and mutant (TopIIIαP1) TRR complexes with 20 nM BLM at 37°C in 30 min. TRR was included in reactions at increasing concentrations ranging from 20 pM to 10 nM. A labeled circular oligonucleotide (lane 1) and dHJ (lane 2) were included as markers. (F) Quantification of the dHJ dissolution reaction shown in (E). The reaction was repeated twice more with very similar results.
In most subjects, the frameshift mutations would be expected to result in nonsense-mediated decay of TOP3A transcripts, explaining the consequent loss of TopIIIα. However, the homozygous frameshift mutation in P1 (c.2718del [p.Thr907LeufsTer101]) is at the 3′ end of the gene and is not predicted to result in NMD; instead, it would result in a protein with a length similar to that of the WT enzyme. The frameshift did, however, result in abolition of the C-terminal zinc-finger domain, whose precise cellular function remains to be defined. We therefore expressed recombinant TopIIIαThr907LeufsTer101 in E. coli and purified it to homogeneity as a complex with the co-expressed RMI1 and RMI2 in order to characterize it further. Notably, in contrast to the TRR complex containing TopIIIαWT, the complex containing TopIIIαThr907LeufsTer101 (hereafter referred to as TopIIIαP1) exhibited reduced stability during purification; lower yields and increased amounts of degraded products were evident on SDS-PAGE (Figure 2C). Nevertheless, when used in quantities equimolar to those of TopIIIαWT, TopIIIαP1 was proficient in a biochemical assay for dHJ dissolution when combined with other components of the BTRR complex. This indicates that this TopIIIα variant retains a near-normal level of single-stranded DNA (ssDNA) decatenation activity (Figures 2D–2F). Therefore, we concluded that the major consequence of the TopIIIαP1 variant, like the other truncating variants, is severe depletion of TopIIIα enzymatic activity in cells.
P5 was the only individual we identified to have an amino acid substitution, p.Ala176Val, that was present in trans with a frameshift mutation. This amino acid substitution is absent from GnomAD, and in silico analyses (MutationTaster, CADD, and SIFT) predict it to be deleterious. It is at a highly conserved residue (Figure S1) within the TOPA domain (Figure 2A) and would therefore be expected to be highly deleterious to enzymatic function.
Given the above results and that Top3a is a developmentally essential gene in mice,34 we conclude that all of the identified mutations result in marked but most likely hypomorphic loss of function of TopIIIα as a result of a reduction in the cellular amount of the protein. Therefore, these mutations would be predicted to severely compromise the decatenation activity of the BTRR complex in dHJ dissolution in vivo. To assess this possibility, we next pursued cellular assays to assess SCE frequency.