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, 287 (16), 13040-7

DNA Lesion Alters Global Conformational Dynamics of Y-family DNA Polymerase During Catalysis

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DNA Lesion Alters Global Conformational Dynamics of Y-family DNA Polymerase During Catalysis

Brian A Maxwell et al. J Biol Chem.

Abstract

A major product of oxidative damage to DNA, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), can lead to genomic mutations if it is bypassed unfaithfully by DNA polymerases in vivo. However, our pre-steady-state kinetic studies show that DNA polymerase IV (Dpo4), a prototype Y-family enzyme from Sulfolobus solfataricus, can bypass 8-oxoG both efficiently and faithfully. For the first time, our stopped-flow FRET studies revealed that a DNA polymerase altered its synchronized global conformational dynamics in response to a DNA lesion. Relative to nucleotide incorporation into undamaged DNA, three of the four domains of Dpo4 undertook different conformational transitions during 8-oxoG bypass and the subsequent extension step. Moreover, the rapid translocation of Dpo4 along DNA induced by nucleotide binding was significantly hindered by the interactions between the embedded 8-oxoG and Dpo4 during the extension step. These results unprecedentedly demonstrate that a Y-family DNA polymerase employs different global conformational dynamics when replicating undamaged and damaged DNA.

Figures

FIGURE 1.
FIGURE 1.
Structure and dynamics of Dpo4 during 8-oxoG bypass and extension steps. The domains of Dpo4 are shown in blue (finger), red (palm), green (thumb), and purple (LF). The DNA is shown in gold. The positions of the Alexa Fluor 594 acceptor on Dpo4 and the Alexa Fluor 488 donor on DNA are shown as yellow and cyan spheres, respectively. The arrows indicate the directions of residue movement during dCTP incorporation opposite 8-oxoG (A) and the subsequent extension of the 8-oxoG bypass product (B) based on the FRET signal changes for phase P1 (black) and phase P2 (white).
FIGURE 2.
FIGURE 2.
Steady-state fluorescence spectra of Dpo4 labeled at LF domain K329C at 20 °C. Alexa Fluor 488-labeled DNA (100 nm; solid black line) was excited at 493 nm. The sequential addition of Alexa Fluor 594-labeled Dpo4 (600 nm) and dTTP (1 mm) produced the dotted gray and dashed black lines, respectively. Spectra were normalized to one by using the donor in the absence of the acceptor as a reference. Emission spectra are shown for both O-2 (A) and O-3 (B) DNA substrates (Table 1).
FIGURE 3.
FIGURE 3.
Stopped-flow traces for dCTP incorporation and binding opposite 8-oxoG at 20 °C. A preincubated solution containing Alexa Fluor 594-labeled Dpo4 (600 nm) and either extendable DNA substrate O-2 (A–D) or non-extendable DNA substrate O-3 (E–H) (100 nm) (Table 1) was rapidly mixed with dCTP (1 mm), and the resulting donor and acceptor fluorescence signals upon excitation of the donor at 493 nm were recorded in real-time. Donor and acceptor traces are shown for the finger domain mutant N70C (A and E), the palm domain mutant S112C (B and F), the thumb domain mutant S207C (C and G), and the LF domain mutant K329C (D and H).
FIGURE 4.
FIGURE 4.
Stopped-flow traces for dGTP incorporation during subsequent extension of 8-oxoG bypass product at 20 °C. A preincubated solution of Alexa Fluor 594-labeled Dpo4 (600 nm) and either extendable DNA substrate O-4 (A–D) or non-extendable DNA substrate O-5 (E–H) (100 nm) (Table 1) was rapidly mixed with dGTP (1 mm), and the resulting donor and acceptor fluorescence signals upon excitation of the donor at 493 nm were recorded over time. Donor and acceptor traces are shown for the finger domain mutant N70C (A and E), the palm domain mutant S112C (B and F), the thumb domain mutant S207C (C and G), and the LF domain mutant K329C (D and H).

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