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The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis

View Article: PubMed Central - PubMed

ABSTRACT

Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B- breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B- tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of ‘APOBEC signature' mutations in cancer.

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A3H haplotype I has enzymatic activity against viral DNA.(a) HIV-1 infectivity assay. Viral and A3 expression vectors are transfected into 293 producer cells and, after 48 h, virus-containing supernatants are titered by infecting CEM-GFP reporter cells in which an integrated LTR-GFP cassette is activated by the Tat protein expressed from newly integrated viruses. Infectivity is quantified by flow cytometry and calculating the percentage of GFP-positive reporter cells. (b) Mean and s.e.m. plotted for three biological replicates of HIV-1 infectivity data for Vif-deficient viruses produced in 293 cells expressing a vector control, A3G-HA, A3H-I or A3H-II (*P<0.05, **P<0.01, ***P<0.001, Welch's two sided t-test). Immunoblots for the indicated proteins in cell lysates and virus containing supernatants are shown below. (c) C-to-T mutation distribution in viral DNA sequences recovered from CEM-GFP reporter cells. C-to-T, C-to-G and C-to-A are represented by red, black and blue shading, respectively. The mutations are reported for the viral cDNA strand, rather than the conventional genomic strand to facilitate comparisons with tumours.
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f4: A3H haplotype I has enzymatic activity against viral DNA.(a) HIV-1 infectivity assay. Viral and A3 expression vectors are transfected into 293 producer cells and, after 48 h, virus-containing supernatants are titered by infecting CEM-GFP reporter cells in which an integrated LTR-GFP cassette is activated by the Tat protein expressed from newly integrated viruses. Infectivity is quantified by flow cytometry and calculating the percentage of GFP-positive reporter cells. (b) Mean and s.e.m. plotted for three biological replicates of HIV-1 infectivity data for Vif-deficient viruses produced in 293 cells expressing a vector control, A3G-HA, A3H-I or A3H-II (*P<0.05, **P<0.01, ***P<0.001, Welch's two sided t-test). Immunoblots for the indicated proteins in cell lysates and virus containing supernatants are shown below. (c) C-to-T mutation distribution in viral DNA sequences recovered from CEM-GFP reporter cells. C-to-T, C-to-G and C-to-A are represented by red, black and blue shading, respectively. The mutations are reported for the viral cDNA strand, rather than the conventional genomic strand to facilitate comparisons with tumours.

Mentions: Next, HIV-1 restriction and mutation assays were used as biological read-outs for A3H-I activity (schematic in Fig. 4a). Vif-deficient HIV-1 particles were produced in 293T cells with a range of untagged A3H concentrations up to maximally tolerated amounts. As expected1314, A3H-II and A3G-HA caused strong dose-responsive decreases in virus infectivity (Fig. 4b). In comparison, overexpression of A3H-I caused more modest, but still significant, drops in virus infectivity (200 ng, P=0.055; 400 ng, P=0.0059, Welch's t-test). These virus restriction phenotypes correlated with the overall amounts of A3H and A3G-HA proteins expressed in cells and packaged into nascent viral particles (Fig. 4b). It is notable that the highest quantity of A3H-I yielded an HIV-1 restriction phenotype similar to the lowest amount of A3H-II, and that these transfected amounts produced similar steady-state protein levels by immunoblotting (red boxed data in Fig. 4b). These HIV-1 restriction phenotypes lend further support to the in vitro biochemical results above and to the surprising finding that A3H-I elicits a level of catalytic activity similar to the better-expressed A3H-II enzyme.


The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis
A3H haplotype I has enzymatic activity against viral DNA.(a) HIV-1 infectivity assay. Viral and A3 expression vectors are transfected into 293 producer cells and, after 48 h, virus-containing supernatants are titered by infecting CEM-GFP reporter cells in which an integrated LTR-GFP cassette is activated by the Tat protein expressed from newly integrated viruses. Infectivity is quantified by flow cytometry and calculating the percentage of GFP-positive reporter cells. (b) Mean and s.e.m. plotted for three biological replicates of HIV-1 infectivity data for Vif-deficient viruses produced in 293 cells expressing a vector control, A3G-HA, A3H-I or A3H-II (*P<0.05, **P<0.01, ***P<0.001, Welch's two sided t-test). Immunoblots for the indicated proteins in cell lysates and virus containing supernatants are shown below. (c) C-to-T mutation distribution in viral DNA sequences recovered from CEM-GFP reporter cells. C-to-T, C-to-G and C-to-A are represented by red, black and blue shading, respectively. The mutations are reported for the viral cDNA strand, rather than the conventional genomic strand to facilitate comparisons with tumours.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: A3H haplotype I has enzymatic activity against viral DNA.(a) HIV-1 infectivity assay. Viral and A3 expression vectors are transfected into 293 producer cells and, after 48 h, virus-containing supernatants are titered by infecting CEM-GFP reporter cells in which an integrated LTR-GFP cassette is activated by the Tat protein expressed from newly integrated viruses. Infectivity is quantified by flow cytometry and calculating the percentage of GFP-positive reporter cells. (b) Mean and s.e.m. plotted for three biological replicates of HIV-1 infectivity data for Vif-deficient viruses produced in 293 cells expressing a vector control, A3G-HA, A3H-I or A3H-II (*P<0.05, **P<0.01, ***P<0.001, Welch's two sided t-test). Immunoblots for the indicated proteins in cell lysates and virus containing supernatants are shown below. (c) C-to-T mutation distribution in viral DNA sequences recovered from CEM-GFP reporter cells. C-to-T, C-to-G and C-to-A are represented by red, black and blue shading, respectively. The mutations are reported for the viral cDNA strand, rather than the conventional genomic strand to facilitate comparisons with tumours.
Mentions: Next, HIV-1 restriction and mutation assays were used as biological read-outs for A3H-I activity (schematic in Fig. 4a). Vif-deficient HIV-1 particles were produced in 293T cells with a range of untagged A3H concentrations up to maximally tolerated amounts. As expected1314, A3H-II and A3G-HA caused strong dose-responsive decreases in virus infectivity (Fig. 4b). In comparison, overexpression of A3H-I caused more modest, but still significant, drops in virus infectivity (200 ng, P=0.055; 400 ng, P=0.0059, Welch's t-test). These virus restriction phenotypes correlated with the overall amounts of A3H and A3G-HA proteins expressed in cells and packaged into nascent viral particles (Fig. 4b). It is notable that the highest quantity of A3H-I yielded an HIV-1 restriction phenotype similar to the lowest amount of A3H-II, and that these transfected amounts produced similar steady-state protein levels by immunoblotting (red boxed data in Fig. 4b). These HIV-1 restriction phenotypes lend further support to the in vitro biochemical results above and to the surprising finding that A3H-I elicits a level of catalytic activity similar to the better-expressed A3H-II enzyme.

View Article: PubMed Central - PubMed

ABSTRACT

Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B- breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B- tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of &lsquo;APOBEC signature' mutations in cancer.

No MeSH data available.


Related in: MedlinePlus