Error-Free Bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Abstract

As one of the most common forms of oxidative DNA damage, 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) generally leads to G:C to T:A mutagenesis. To study DNA replication encountering 8-oxoG by the sole DNA polymerase (Gp90) of Pseudomonas aeruginosa phage PaP1, we performed steady-state and pre-steady-state kinetic analyses of nucleotide incorporation opposite 8-oxoG by Gp90 D234A that lacks exonuclease activities on ssDNA and dsDNA substrates. Gp90 D234A could bypass 8-oxoG in an error-free manner, preferentially incorporate dCTP opposite 8-oxoG, and yield similar misincorporation frequency to unmodified G. Gp90 D234A could extend beyond C:8-oxoG or A:8-oxoG base pairs with the same efficiency. dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG showed fast burst phases. The burst of incorporation efficiency (kpol/Kd,dNTP) is decreased as dCTP:G > dCTP:8-oxoG > dATP:8-oxoG. The presence of 8-oxoG in DNA does not affect its binding to Gp90 D234A in a binary complex but it does affect it in a ternary complex with dNTP and Mg2+, and dATP misincorporation opposite 8-oxoG further weakens the binding of Gp90 D234A to DNA. This study reveals Gp90 D234A can bypass 8-oxoG in an error-free manner, providing further understanding in DNA replication encountering oxidation lesion for P.aeruginosa phage PaP1.

Protein Interactions in the T7 DNA Replisome Facilitate DNA Damage Bypass

“…[40] The T4 DNA holoenzyme cannotb ypass al ocked nucleic acid (lesion)b ut it can overcome this lesion in Figure 7. [42] Thus, the accessory proteins in other DNA replisomes also facilitate the DNA damage bypass. The DNA replicationfork (30-mer/ 63-mer/62-mer,3 00 RU) containingG ,8 -oxoG, or O 6 -MeG was coupled to an SA chip.…”

“…Most significantly,M cm10 enables CMG and the replisome to bypass dual streptavidin blocks on the laggingD NA strand. [42] Thus, the accessory proteins in other DNA replisomes also facilitate the DNA damage bypass.…”

“…[2,42] Strand-displacement DNA synthesis was initiated by rapidly mixing 32 P-labeled 27-mer/63-mer/62-mer ("S") or 32 Plabeled 20-mer/70-mer/62-mer ("R") fork DNA substrate (40 nm) containing G, 8-oxoG, or O 6 -MeG, gp5/trx (40 nm), gp4 or gp4 DCtail (monomer concentration, 2.16 mm), and gp2.5 (0 or 24 mm) with the four dNTPs (300 mm)i nb uffer Aa t3 78C. [2,42] Strand-displacement DNA synthesis was initiated by rapidly mixing 32 P-labeled 27-mer/63-mer/62-mer ("S") or 32 Plabeled 20-mer/70-mer/62-mer ("R") fork DNA substrate (40 nm) containing G, 8-oxoG, or O 6 -MeG, gp5/trx (40 nm), gp4 or gp4 DCtail (monomer concentration, 2.16 mm), and gp2.5 (0 or 24 mm) with the four dNTPs (300 mm)i nb uffer Aa t3 78C.…”

“…Strand-displacement DNA synthesis past DNA damage by polymerase and helicase complex:R apid quench experiments were performed with am odel RQF-3 KinTek Quench Flow Apparatus (KinTek Corp, Austin, TX, USA) with Tris·HCl buffer (50 mm,p H7.5) in both drive syringes and EDTAs olution (0.5 m)i nt he middle quench syringe. [2,42] Strand-displacement DNA synthesis was initiated by rapidly mixing 32 P-labeled 27-mer/63-mer/62-mer ("S") or 32 Plabeled 20-mer/70-mer/62-mer ("R") fork DNA substrate (40 nm) containing G, 8-oxoG, or O 6 -MeG, gp5/trx (40 nm), gp4 or gp4 DCtail (monomer concentration, 2.16 mm), and gp2.5 (0 or 24 mm) with the four dNTPs (300 mm)i nb uffer Aa t3 78C. After the indicated times, the reactions were rapid quenched with EDTA( 0.5 m).…”

Erratum: Gu, S. et al. Error-Free Bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1. Genes 2017, 8, 18

“…The authors wish to make the following correction to their paper [1]. The title of the paper should be corrected to “Error-Free Bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1”.…”