Structural and Dynamic Characterization of Polymerase κ’s Minor Groove Lesion Processing Reveals How Adduct Topology Impacts Fidelity

DNA lesion bypass polymerases process different lesions with varying fidelities, but the structural, dynamic, and mechanistic origins of this phenomenon remain poorly understood. Human DNA polymerase κ (Polκ), a member of the Y family of lesion bypass polymerases, is specialized to bypass bulky DNA minor groove lesions in a predominantly error-free manner, by housing them in its unique gap. We have investigated the role of the unique Polκ gap and N-clasp structural features in the fidelity of minor groove lesion processing with extensive molecular modeling and molecular dynamics simulations to pinpoint their functioning in lesion bypass. Here we consider the <i>N</i>²-dG covalent adduct derived from the carcinogenic aromatic amine, 2-acetylaminofluorene (dG-<i>N</i>²-AAF), that is produced via the combustion of kerosene and diesel fuel. Our simulations reveal how the spacious gap directionally accommodates the lesion aromatic ring system as it transits through the stages of incorporation of the predominant correct partner dCTP opposite the damaged guanine, with preservation of local active site organization for nucleotidyl transfer. Furthermore, flexibility in Polκ’s N-clasp facilitates the significant misincorporation of dTTP opposite dG-<i>N</i>²-AAF via wobble pairing. Notably, we show that N-clasp flexibility depends on lesion topology, being markedly reduced in the case of the benzo­[<i>a</i>]­pyrene-derived major adduct to <i>N</i>²-dG, whose bypass by Polκ is nearly error-free. Thus, our studies reveal how Polκ’s unique structural and dynamic properties can regulate its bypass fidelity of polycyclic aromatic lesions and how the fidelity is impacted by lesion structures

N‑Geminal P/Al Lewis Pair–Alkyne Dipolar Cycloaddition to the Zwitterionic C₂PNAl-Heterocyclopentene

The N-geminal P/Al Lewis pair [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]₂ (<b>1</b>) has been prepared and studied for reaction with a series of alkynes. The reaction of <b>1</b> with RCCR yielded zwitterionic C₂PNAl-heterocyclopentene [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]­(CRCR) (R = Me (<b>2</b>), Ph (<b>3</b>)); with PhCCEt produced two isomers, [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]­(CPhCEt) (<b>4a</b>) and [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]­(CEt­CPh) (<b>4b</b>); and with other alkynes generated [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]­(CR¹CR²) (R¹, R² = CO₂Et, Ph (<b>5</b>); SiMe₃, Ph (<b>6</b>); PPh₂, Ph (<b>7</b>); SiMe₃,H (<b>8</b>); H, EtO (<b>9</b>)). Natural bond orbital analysis of the charge separation of the CC bond of alkynes was carried out, and then, the electronic matching interaction mode between the combined Lewis acid (AlEt₂) and base (PPh₂) groups of <b>1</b> and the CC bond of such alkynes was discussed. Reactions of <b>1</b> with alkene, nitrile, and carbodiimide molecules were also carried out, and cycloaddition compounds <b>10</b>–<b>12</b> were produced

1D and 2D NMR spectra characteristics of the <i>trans</i>-B[<i>a</i>]P-dG:AB duplex.

<p>(A) 1D spectrum (10.5–14 ppm) showing the imino proton assignments. (B) Portion of a 2D NOESY (250 ms mixing time) contour plot recorded at 10°C, in 10% H₂O solution showing NOE connectivities between amino (5–8.5 ppm) and imino protons (10.8–13.8 ppm), and (C) imino- imino protons (10.8–13.8 ppm). Assignments: a, T20(NH3)—G21(NH1); b, T14(NH3)—G13(NH1); c, T4(NH3)—G18(NH1); d, T8(NH3)—G16(NH1); e, A3(H2)—T20(NH3); f, A9(H2)—T14(NH3); g, A19(H2)—T4(NH3); h, A15(H2)—T8(NH3); i,i’, C2(NH,H’)—G21(NH1); j, C2(H5)—G21(NH1); k, k’, C10(NH,H’)—G13(NH1); <i>l</i>, C10(H5)—G13(NH1); m, m’, C5(NH,H’)—G18(NH1); n, C5(H5)—G18(NH1); o, o’, C7(NH,H’)—G16(NH1); p, C7(H5)—G(NH1); q, A3(H2)—G21(NH1); r, A9(H2)—G13(NH1); s, A19(H2)—G18(NH1); t, A15(H2)—G16(NH1).</p

N‑Geminal P/Al Lewis Pair–Alkyne Dipolar Cycloaddition to the Zwitterionic C₂PNAl-Heterocyclopentene

The N-geminal P/Al Lewis pair [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]₂ (<b>1</b>) has been prepared and studied for reaction with a series of alkynes. The reaction of <b>1</b> with RCCR yielded zwitterionic C₂PNAl-heterocyclopentene [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]­(CRCR) (R = Me (<b>2</b>), Ph (<b>3</b>)); with PhCCEt produced two isomers, [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]­(CPhCEt) (<b>4a</b>) and [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]­(CEt­CPh) (<b>4b</b>); and with other alkynes generated [Ph₂PN­(2,6-<i>i</i>Pr₂C₆H₃)­AlEt₂]­(CR¹CR²) (R¹, R² = CO₂Et, Ph (<b>5</b>); SiMe₃, Ph (<b>6</b>); PPh₂, Ph (<b>7</b>); SiMe₃,H (<b>8</b>); H, EtO (<b>9</b>)). Natural bond orbital analysis of the charge separation of the CC bond of alkynes was carried out, and then, the electronic matching interaction mode between the combined Lewis acid (AlEt₂) and base (PPh₂) groups of <b>1</b> and the CC bond of such alkynes was discussed. Reactions of <b>1</b> with alkene, nitrile, and carbodiimide molecules were also carried out, and cycloaddition compounds <b>10</b>–<b>12</b> were produced

Expanded contour plot of a NOESY spectrum (300 ms mixing time) of the <i>trans-</i>B[<i>a</i>]P-dG:AB duplex.

<p>The spectrum was obtained in D₂O aqueous buffer solution at 10°C using an 800 MHz spectrometer equipped with a cryoprobe. The focus is on the base (purine H8 and pyrimidine H5 and H6) and sugar H1’ proton region. (A) Sequential assignments from dC1 to dC11 on the modified strand (blue dashed line) A:B[<i>a</i>]P10-B[<i>a</i>]P11, B: B[<i>a</i>]P10-B[<i>a</i>]P12, C: B[<i>a</i>]P9-B[<i>a</i>]P11, D: B[<i>a</i>]P8-B[<i>a</i>]P11, E: B[<i>a</i>]P7-B[<i>a</i>]P6, F: B[<i>a</i>]P7-B[<i>a</i>]P5; 1: G6(H1’)-B[<i>a</i>]P12, 2: C7(H5)-B[<i>a</i>]P12, 3: C5(H1’)-B[<i>a</i>]P12, 4: G6(H1’)-B[<i>a</i>]P11, 5: C7(H5)-B[<i>a</i>]P11, 6: G6(H1’)-B[<i>a</i>]P10, 7: C7(H5)-B[<i>a</i>]P10, 8: G6(H3’)-H8, 9: G6(H3’)-C7(H6), 10: G6(H3’)-(H1’), 11: C7(H5)-G6(H1’); a: C10(H5)-(H6), b: C11(H5)-(H6), c: C2(H5)-(H6), d: C7(H5)-(H6), e: C1(H5)-(H6) (B) Sequential assignments from dG12 to dG16 (purple dashed line); from dG18 to dG22 (black dashed line) in the THF-containing complementary strand. Expanded NOESY contour plot in D₂O buffer at 10°C establishing the NOE connectivity between base protons (purine H8 and pyrimidine H6) and their own and 5’-flanking sugar H1’ protons.</p

Chemical structures of the lesions and sequences.

<p>(A) Chemical structures of the <i>trans</i>-B[<i>a</i>]P-dG, <i>cis</i>-B[<i>a</i>]P-dG and the THF site. The THF is a stable analog of an abasic (AB) site. (B) The sequences and numbering system of the 11-mer duplexes containing the <i>trans</i>-B[<i>a</i>]P-dG adduct. G* denotes B[<i>a</i>]P-dG adduct.</p

UV absorption spectra differ with intercalated and non-intercalated aromatic ring systems.

<p>Titration curves of 11-mer strands containing <i>trans-</i>B[<i>a</i>]P-dG (A and B) or <i>cis</i>-B[<i>a</i>]P-dG (C and D) adducts with full complementary 11-mer strands (A and C) or 11-mer strands with abasic THF sites (B and D). The heavy black lines represent the initial absorption spectra of the modified single-stranded oligonucleotides, while the final spectra of the fully titrated duplex end points are denoted by heavy red lines. The intermediate grey lines represent absorption spectra measured at intermediate titration points that correspond to successive additions of aliquots of the complementary strands. Each aliquot contained a 10% concentration relative to that of the fixed concentration of the modified strand. The initial concentrations of the modified strand are ~ 5 μM.</p

Comparison of chemical shifts of B[<i>a</i>]P protons: <i>trans</i>-B[<i>a</i>]P-dG:AB 11/11-mer duplex (□); <i>trans</i>-B[<i>a</i>]P-dG:dC duplex in a full, normally base-paired 11/11mer duplex with the aromatic pyrenyl residue in the minor grove [24] (●); stereoisomeric <i>cis-</i>B[<i>a</i>]P-dG:dC 11/11-mer duplex [22] (▲); <i>trans</i>-B[<i>a</i>]P-dG:del 11/10-mer duplex [18] (▽).

<p>Comparison of chemical shifts of B[<i>a</i>]P protons: <i>trans</i>-B[<i>a</i>]P-dG:AB 11/11-mer duplex (□); <i>trans</i>-B[<i>a</i>]P-dG:dC duplex in a full, normally base-paired 11/11mer duplex with the aromatic pyrenyl residue in the minor grove [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137124#pone.0137124.ref024" target="_blank">24</a>] (●); stereoisomeric <i>cis-</i>B[<i>a</i>]P-dG:dC 11/11-mer duplex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137124#pone.0137124.ref022" target="_blank">22</a>] (▲); <i>trans</i>-B[<i>a</i>]P-dG:del 11/10-mer duplex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137124#pone.0137124.ref018" target="_blank">18</a>] (▽).</p

Time course of NER and BER excision cleavage product formation.

<p>(A) Time course of cleavage of the DNA strand with the abasic THF site opposite G in the opposite strand either without (unmodified), or with a <i>trans</i>- or a <i>cis-</i>B[<i>a</i>]P-dG adduct (abbreviated by a “B” in this Figure) opposite the AB site in the complementary strand. The solid lines are plots of the standard base excision repair burst equation P = A(1—exp[-<i>k</i>t]) + k_ss t. The values of A are 81, 65 and 41% for the unmodified, <i>trans</i>-B[<i>a</i>]P-dG, and <i>cis</i>-B[<i>a</i>]P-dG adduct-containing complementary strands, with single turnover constants <i>k</i> = 5.0, 5.0, and 0.50 min^-1, and the steady-state constant <i>k</i>_<i>ss</i> = 0, 0.33 and 0.43 min^-1, respectively. (B) Time course of NER dual incision product formation. All data points were obtained from densitometry analyses of the gel shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137124#pone.0137124.g007" target="_blank">Fig 7</a>.</p

Thermal melting, conformation and repair data for B[<i>a</i>]P-dG adducts and sequences investigated.

<p>^<i>a</i> ΔT_m = T_m(<i>modified</i>)—T_m(<i>unmodified</i>)</p><p>Thermal melting, conformation and repair data for B[<i>a</i>]P-dG adducts and sequences investigated.</p