Limits...
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.

No MeSH data available.


A3H haplotype I is an active DNA cytosine deaminase.(a) Schematic of the ssDNA deamination assay. A3H-mediated deamination yields a uracil that, on excision by excess uracil DNA glycosylase, is converted into a hydroxide-labile abasic site. (b) Anti-FLAG immunoblot of A3H-I, A3H-II and catalytic mutant derivatives expressed in 293T cells prior to purification, with an anti-ACTB immunoblot shown below as a loading control. (c) Image of a Coomassie-stained gel with approximately equal amounts of A3H-I, A3H-II and catalytic mutant derivatives purified from 293T cells. (d) Activity data for the recombinant A3H proteins shown in c (P, product; S, substrate). (e) Activity of GST-A3H-I purified from insect cells using the indicated trinucleotide containing ssDNA substrates (P, product; S, substrate).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036005&req=5

f3: A3H haplotype I is an active DNA cytosine deaminase.(a) Schematic of the ssDNA deamination assay. A3H-mediated deamination yields a uracil that, on excision by excess uracil DNA glycosylase, is converted into a hydroxide-labile abasic site. (b) Anti-FLAG immunoblot of A3H-I, A3H-II and catalytic mutant derivatives expressed in 293T cells prior to purification, with an anti-ACTB immunoblot shown below as a loading control. (c) Image of a Coomassie-stained gel with approximately equal amounts of A3H-I, A3H-II and catalytic mutant derivatives purified from 293T cells. (d) Activity data for the recombinant A3H proteins shown in c (P, product; S, substrate). (e) Activity of GST-A3H-I purified from insect cells using the indicated trinucleotide containing ssDNA substrates (P, product; S, substrate).

Mentions: A3H-I, A3H-II and catalytic mutant derivatives (E56A) were purified from human 293T cells, normalized to be equimolar and assayed for DNA cytosine deaminase activity using a gel-based ssDNA deamination assay727325556 (schematic in Fig. 3a). In agreement with prior studies4647484951, A3H-I showed lower overall protein expression levels in cellular lysates in comparison to A3H-II (Fig. 3b). However, similar concentrations of each protein could be achieved by concentrating A3H-I, as evidenced by near-equivalent A3H band intensities on direct visualization of SDS–polyacrylamide gel electrophoresis fractionated proteins (Fig. 3c). A head-to-head comparison of A3H-I and A3H-II using a ssDNA with a single TCA target motif indicated near-equivalent enzymatic activities (Fig. 3d). Importantly, the E56A catalytic mutant derivatives purified and analysed in parallel had no detectable catalytic activity, which demonstrates that all of the observed activity is due to active A3H-I or A3H-II (and not, for instance, to a co-purifying factor from 293T cells). In independent experiments, recombinant A3H-I from Sf9 insect cells also elicited ssDNA C-to-U editing activity indicating no other human factors are required (Fig. 3e).


The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis
A3H haplotype I is an active DNA cytosine deaminase.(a) Schematic of the ssDNA deamination assay. A3H-mediated deamination yields a uracil that, on excision by excess uracil DNA glycosylase, is converted into a hydroxide-labile abasic site. (b) Anti-FLAG immunoblot of A3H-I, A3H-II and catalytic mutant derivatives expressed in 293T cells prior to purification, with an anti-ACTB immunoblot shown below as a loading control. (c) Image of a Coomassie-stained gel with approximately equal amounts of A3H-I, A3H-II and catalytic mutant derivatives purified from 293T cells. (d) Activity data for the recombinant A3H proteins shown in c (P, product; S, substrate). (e) Activity of GST-A3H-I purified from insect cells using the indicated trinucleotide containing ssDNA substrates (P, product; S, substrate).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036005&req=5

f3: A3H haplotype I is an active DNA cytosine deaminase.(a) Schematic of the ssDNA deamination assay. A3H-mediated deamination yields a uracil that, on excision by excess uracil DNA glycosylase, is converted into a hydroxide-labile abasic site. (b) Anti-FLAG immunoblot of A3H-I, A3H-II and catalytic mutant derivatives expressed in 293T cells prior to purification, with an anti-ACTB immunoblot shown below as a loading control. (c) Image of a Coomassie-stained gel with approximately equal amounts of A3H-I, A3H-II and catalytic mutant derivatives purified from 293T cells. (d) Activity data for the recombinant A3H proteins shown in c (P, product; S, substrate). (e) Activity of GST-A3H-I purified from insect cells using the indicated trinucleotide containing ssDNA substrates (P, product; S, substrate).
Mentions: A3H-I, A3H-II and catalytic mutant derivatives (E56A) were purified from human 293T cells, normalized to be equimolar and assayed for DNA cytosine deaminase activity using a gel-based ssDNA deamination assay727325556 (schematic in Fig. 3a). In agreement with prior studies4647484951, A3H-I showed lower overall protein expression levels in cellular lysates in comparison to A3H-II (Fig. 3b). However, similar concentrations of each protein could be achieved by concentrating A3H-I, as evidenced by near-equivalent A3H band intensities on direct visualization of SDS–polyacrylamide gel electrophoresis fractionated proteins (Fig. 3c). A head-to-head comparison of A3H-I and A3H-II using a ssDNA with a single TCA target motif indicated near-equivalent enzymatic activities (Fig. 3d). Importantly, the E56A catalytic mutant derivatives purified and analysed in parallel had no detectable catalytic activity, which demonstrates that all of the observed activity is due to active A3H-I or A3H-II (and not, for instance, to a co-purifying factor from 293T cells). In independent experiments, recombinant A3H-I from Sf9 insect cells also elicited ssDNA C-to-U editing activity indicating no other human factors are required (Fig. 3e).

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.

No MeSH data available.