Replication past the N5-methyl-formamidopyrimidine lesion of deoxyguanosine by DNA polymerases and an improved procedure for sequence analysis of in vitro bypass products by mass spectrometry

Abstract

Oligonucleotides containing a site-specific N(6)-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) lesion were synthesized, and their in vitro replication by Escherichia coli DNA polymerase I Klenow fragment (exo(-)) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) resulted in the misincorporation of Ade, Gua, and Thy opposite the MeFapy-dGuo lesion in addition to the correct insertion of Cyt. However, sequencing of the full-length extension products revealed that the initial insertion of Cyt opposite the lesion was extended most efficiently. Two sequences were examined, and the misincorporation was sequence-dependent. Improvements in the method for the mass spectrometric sequencing of the extension products were developed; a 5'-biotinylated primer strand was used that contained a dUrd near the template-primer junction. The extended primer was immobilized with streptavidin-coated beads, allowing it to be washed free of polymerase, the template strand, and other reagents. The extended primer was cleaved from the solid support with uridine DNA deglycosylase and piperidine treatment, and the extension products were sequenced by LC-ESI-MS-MS. The purification steps afforded by the biotinylated primer resulted in improved sensitivity for the MS analysis. Translesion synthesis of a template with a local 5'-T-(MeFapy-dGuo)-G-3' sequence resulted in only error-free bypass and extension, whereas a template with a local 5'-T-(MeFapy-dGuo)-T-3' sequence also resulted in an interesting deletion product and the misincorporation of Ade opposite the MeFapy-dGuo lesion.

Figure 1

Bypass and extension of the MeFapy-dGuo lesion by Kf⁻ and Dpo4 with increasing concentration of dNTPs (µM).

Figure 2

UPLC-ESI-MS-MS analysis of the extension of the 5'-T-(MeFapy-dGuo)-G-3' template 1 by Kf⁻. A. UPLC trace of the extension reaction vs. total ion current. B. Full-scan product ion spectrum of extension reaction products with an internal standard. C. CID product ion spectrum of the extension product (m/z=1078.7 Da.) D. CID spectrum of an authentic standard of the extension product.

Figure 3

UPLC-ESI-MS-MS analysis of the extension of the 5'-T-(MeFapy-dGuo)-T-3' template 2 by Dpo4. A. Extracted ion profile of the sum of the extracted [M-2H] and [M-3H] ions extension products and [M-3H] and [M-4H] of the standard. B. Full-scan mass spectrum of extension reaction products at retention time 4.75 min with an internal standard. C. CID spectrum of the extension product 5'-pTGAC–CGA-3' (m/z = 1091.5) D. CID spectrum of an authentic standard of the extension product.

Figure 4

Averaged full scan spectrum from t = 3.90 – 4.20 min (A) and the extracted ion profile (B) of a known mixture of the five extension products (sum of the M-2H] and [M-3H] ions) from of the 5'-T-(MeFapy-dGuo)-T-3' template (2) with Kf⁻ and the 13mer standard (sum of the [M-3H] and [M-4H] ions).

Figure 5

Models for the instructive insertion of dCTP and dATP opposite Fapy-dGuo lesions.

Scheme 1

Scheme 2