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Alternative divalent cations (Zn²⁺, Co²⁺, and Mn²⁺) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity.

Achuthan V, DeStefano JJ - BMC Biochem. (2015)

Bottom Line: The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn(2+) when compared to Mg(2+) at cation conditions optimized for nucleotide catalysis.In agreement with previous literature, we observed that Mn(2+) and Co(2+) dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM).This study shows that Zn(2+), at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA. vasuachu@umd.edu.

ABSTRACT

Background: Fidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg(2+) is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn(2+), Co(2+), and Zn(2+) can also support catalysis. Although Zn(2+) supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn(2+) is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn(2+).

Methods: We used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn(2+), Co(2+), and Zn(2+).

Results: The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn(2+) when compared to Mg(2+) at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3' nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn(2+) than Mg(2+). In agreement with previous literature, we observed that Mn(2+) and Co(2+) dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn(2+) and Co(2+) remained similar to Mg(2+) at lower concentrations that are optimal for catalysis.

Conclusion: This study shows that Zn(2+), at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.

No MeSH data available.


DNA sequence analysis from the PCR-based lacZα-complementation fidelity assay. The 115 base region analyzed for mutations is shown. The coding strand for lacZα is shown in the 5-3′ direction (bottom strand in Figure 2C). Numbering is as shown in Figure 2C. Deletions are shown as regular triangles, insertions are shown as downward triangles with the inserted base shown adjacent to the downward triangle, unless it was the same as the base in a nt run, and base substitutions are shown directly above or below the sequence. Substitutions shown correspond to the recovered sequence for the coding strand; however, these mutations could have occurred during synthesis of the non-coding strand as well (i.e. a C to A change shown here could have resulted from a C to A change during synthesis of the coding strand or a G to T during synthesis of the non-coding strand) (see Figure 2). Mutations recovered from HIV RT with 2 mM Mg2±, and mutations from background controls are shown above the sequence as open triangles and normal text or filled triangles and bold italicized text, respectively. Mutations from HIV RT at 0.4 mM Zn2+ are shown below the sequence. Individual sequence clones which had multiple mutations (more than one mutation event) are marked with subscripts adjacent to the mutations. Several clones with deletions (either single or multiple deletions) at positions 181–183, just outside of the scored region were also recovered (not shown). This was the dominant mutation type recovered in background controls (19 out of 24 total sequences) and probably resulted from improper ligation events or damaged plasmid vectors (see [48]).
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Fig3: DNA sequence analysis from the PCR-based lacZα-complementation fidelity assay. The 115 base region analyzed for mutations is shown. The coding strand for lacZα is shown in the 5-3′ direction (bottom strand in Figure 2C). Numbering is as shown in Figure 2C. Deletions are shown as regular triangles, insertions are shown as downward triangles with the inserted base shown adjacent to the downward triangle, unless it was the same as the base in a nt run, and base substitutions are shown directly above or below the sequence. Substitutions shown correspond to the recovered sequence for the coding strand; however, these mutations could have occurred during synthesis of the non-coding strand as well (i.e. a C to A change shown here could have resulted from a C to A change during synthesis of the coding strand or a G to T during synthesis of the non-coding strand) (see Figure 2). Mutations recovered from HIV RT with 2 mM Mg2±, and mutations from background controls are shown above the sequence as open triangles and normal text or filled triangles and bold italicized text, respectively. Mutations from HIV RT at 0.4 mM Zn2+ are shown below the sequence. Individual sequence clones which had multiple mutations (more than one mutation event) are marked with subscripts adjacent to the mutations. Several clones with deletions (either single or multiple deletions) at positions 181–183, just outside of the scored region were also recovered (not shown). This was the dominant mutation type recovered in background controls (19 out of 24 total sequences) and probably resulted from improper ligation events or damaged plasmid vectors (see [48]).

Mentions: An estimate of the base misincorporation frequency can be made from the CMFs in Table 2 and the sequencing results in Figure 3 as described before [48]. In experiments with Mg2+, ~41% (17/42) of recovered mutations, after excluding the background mutations, were insertions or deletions (indels), and ~59% (25/42) substitutions. Using a 33.6% detection rate for substitutions and 100% detection rate for indels in this region (see Figure 2C and accompanying legend) and a CMF of 0.0059 (from Table 1), the mutation frequency for Mg2+ was 5.6 × 10−5, or ~1 error per 18,000 incorporations ((0.0059 × 0.41)/230 = 1.1 × 10−5 for indels, and ((0.0059 × 0.59)/230)/0.336 = 4.5 × 10−5 for substitutions, total is 5.6 × 10−5 for both (see Figure 3 legend for further details)). Synthesis with Zn2+ resulted in a higher ratio of indels vs. substitution: indels ~63% (26/41), and ~37% substitutions (15/41) were obtained. With a CMF of 0.0025 (Table 1), a mutation frequency of 1.9 × 10−5 or ~ 1 error per 53,000 incorporations was obtained for experiments with Zn2+. This value is also closer to the rate of ~1 error per 77,000 incorporations that was observed with more physiological (0.25 mM), though sub-optimal Mg2+ concentrations [4].Figure 3


Alternative divalent cations (Zn²⁺, Co²⁺, and Mn²⁺) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity.

Achuthan V, DeStefano JJ - BMC Biochem. (2015)

DNA sequence analysis from the PCR-based lacZα-complementation fidelity assay. The 115 base region analyzed for mutations is shown. The coding strand for lacZα is shown in the 5-3′ direction (bottom strand in Figure 2C). Numbering is as shown in Figure 2C. Deletions are shown as regular triangles, insertions are shown as downward triangles with the inserted base shown adjacent to the downward triangle, unless it was the same as the base in a nt run, and base substitutions are shown directly above or below the sequence. Substitutions shown correspond to the recovered sequence for the coding strand; however, these mutations could have occurred during synthesis of the non-coding strand as well (i.e. a C to A change shown here could have resulted from a C to A change during synthesis of the coding strand or a G to T during synthesis of the non-coding strand) (see Figure 2). Mutations recovered from HIV RT with 2 mM Mg2±, and mutations from background controls are shown above the sequence as open triangles and normal text or filled triangles and bold italicized text, respectively. Mutations from HIV RT at 0.4 mM Zn2+ are shown below the sequence. Individual sequence clones which had multiple mutations (more than one mutation event) are marked with subscripts adjacent to the mutations. Several clones with deletions (either single or multiple deletions) at positions 181–183, just outside of the scored region were also recovered (not shown). This was the dominant mutation type recovered in background controls (19 out of 24 total sequences) and probably resulted from improper ligation events or damaged plasmid vectors (see [48]).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig3: DNA sequence analysis from the PCR-based lacZα-complementation fidelity assay. The 115 base region analyzed for mutations is shown. The coding strand for lacZα is shown in the 5-3′ direction (bottom strand in Figure 2C). Numbering is as shown in Figure 2C. Deletions are shown as regular triangles, insertions are shown as downward triangles with the inserted base shown adjacent to the downward triangle, unless it was the same as the base in a nt run, and base substitutions are shown directly above or below the sequence. Substitutions shown correspond to the recovered sequence for the coding strand; however, these mutations could have occurred during synthesis of the non-coding strand as well (i.e. a C to A change shown here could have resulted from a C to A change during synthesis of the coding strand or a G to T during synthesis of the non-coding strand) (see Figure 2). Mutations recovered from HIV RT with 2 mM Mg2±, and mutations from background controls are shown above the sequence as open triangles and normal text or filled triangles and bold italicized text, respectively. Mutations from HIV RT at 0.4 mM Zn2+ are shown below the sequence. Individual sequence clones which had multiple mutations (more than one mutation event) are marked with subscripts adjacent to the mutations. Several clones with deletions (either single or multiple deletions) at positions 181–183, just outside of the scored region were also recovered (not shown). This was the dominant mutation type recovered in background controls (19 out of 24 total sequences) and probably resulted from improper ligation events or damaged plasmid vectors (see [48]).
Mentions: An estimate of the base misincorporation frequency can be made from the CMFs in Table 2 and the sequencing results in Figure 3 as described before [48]. In experiments with Mg2+, ~41% (17/42) of recovered mutations, after excluding the background mutations, were insertions or deletions (indels), and ~59% (25/42) substitutions. Using a 33.6% detection rate for substitutions and 100% detection rate for indels in this region (see Figure 2C and accompanying legend) and a CMF of 0.0059 (from Table 1), the mutation frequency for Mg2+ was 5.6 × 10−5, or ~1 error per 18,000 incorporations ((0.0059 × 0.41)/230 = 1.1 × 10−5 for indels, and ((0.0059 × 0.59)/230)/0.336 = 4.5 × 10−5 for substitutions, total is 5.6 × 10−5 for both (see Figure 3 legend for further details)). Synthesis with Zn2+ resulted in a higher ratio of indels vs. substitution: indels ~63% (26/41), and ~37% substitutions (15/41) were obtained. With a CMF of 0.0025 (Table 1), a mutation frequency of 1.9 × 10−5 or ~ 1 error per 53,000 incorporations was obtained for experiments with Zn2+. This value is also closer to the rate of ~1 error per 77,000 incorporations that was observed with more physiological (0.25 mM), though sub-optimal Mg2+ concentrations [4].Figure 3

Bottom Line: The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn(2+) when compared to Mg(2+) at cation conditions optimized for nucleotide catalysis.In agreement with previous literature, we observed that Mn(2+) and Co(2+) dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM).This study shows that Zn(2+), at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA. vasuachu@umd.edu.

ABSTRACT

Background: Fidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg(2+) is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn(2+), Co(2+), and Zn(2+) can also support catalysis. Although Zn(2+) supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn(2+) is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn(2+).

Methods: We used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn(2+), Co(2+), and Zn(2+).

Results: The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn(2+) when compared to Mg(2+) at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3' nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn(2+) than Mg(2+). In agreement with previous literature, we observed that Mn(2+) and Co(2+) dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn(2+) and Co(2+) remained similar to Mg(2+) at lower concentrations that are optimal for catalysis.

Conclusion: This study shows that Zn(2+), at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.

No MeSH data available.