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

Abstract

Background: Y-family DNA polymerases may employ protein conformational changes to catalyze the bypass of various DNA lesions. Results: The conformational dynamics of three structural domains of Dpo4 are altered by 8-oxo-7,8-dihydro-2Ј-deoxyguanine (8-oxoG). Conclusion: Dpo4 undergoes different global conformational changes during 8-oxoG bypass than during the replication of undamaged DNA. Significance: A DNA lesion alters the conformational dynamics of a DNA polymerase during catalysis.

Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

“…Notably, fluorescence changes are shown on an arbitrary scale and thus the absolute change in FRET efficiency cannot be calculated from these data as it can for the steady-state fluorescence data (Table 2). In contrast to the global conformational dynamics previously observed with Dpo4 by us 29; 30 , the observed FRET phases for mutants of Taq Pol all followed the same trend, indicating that all monitored positions moved in the same direction relative to the DNA substrate. The phases were characterized by a very rapid acceptor fluorescence intensity decrease (P 0 = 11.5–36 s −1 ), followed by a slow increase (P 1 = 0.08–0.26 s −1 ) and then a slower decrease phase (P 2 = 0.006–0.017 s −1 ) (Table 3).…”

“…The rapid FRET decrease (P 0 ) observed for all mutants of Taq Pol was not previously detected for the Finger domain in Klentaq1 17 . However, a similar rapid FRET increase was previously proposed to represent a rapid one base pair translocation of the DNA substrate induced by nucleotide binding to Dpo4 29; 30 . Likewise, P 0 may represent a similar rapid DNA motion in Taq Pol though an additional protein conformational change cannot be ruled out.…”

“…Furthermore, as the rate of P 2 (0.006–0.017 s −1 ) was ~10-fold lower than the rate-limiting step of nucleotide incorporation (0.076–0.136 s −1 ), this conformational change must be limited by a slow process occurring after the chemistry step. Previous studies with Klentaq1 19 or Dpo4 29; 30 have found the analogous post-catalytic motion to be limited only by the rate-limiting step of nucleotide incorporation. Consistently, our kinetic assays here show that the k p values for the Klentaq mutants (0.22–0.33 s −1 , Table S2) are much slower than the P 1 rates (1.7–4.9 s −1 ) and only slightly faster than the P 2 rates (0.05–0.17 s −1 ) (Table 4).…”

“…However, while structural studies of the Y-Family DNA polymerases have revealed no obvious conformational change analogous to the Finger domain closing in other DNA polymerases, our recent studies with Solfolobus Solfataricus DNA polymerase IV (Dpo4), a model Y-Family DNA polymerase, have revealed global conformational changes in all four domains of the enzyme 29; 30 . Additionally, Molecular Dynamics simulations have shown that short distance conformational changes throughout the structure of HIV-1 RT accompany the larger finger domain closing during nucleotide binding 31 .…”

“…Alexa488 (Molecular Probes, Invitrogen) was attached to a 5-C6-Amino-2′-deoxythymidine on the ninth base from the primer 3′-end of the DNA substrates. Alexa488-labeled DNA was purified according to the manufacturer's protocol and annealed as described previously 29; 30 .…”