Following DNA Chain Extension and Protein Conformational Changes in Crystals of a Y-Family DNA Polymerase via Raman Crystallography

Abstract

Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. 13C, 15N labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg2+ or Mn2+, nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of C* modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following single d*CTP addition. Adding these two bases caused visible damage to the crystal possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn2+ or Mg2+, the primer extension reaction did not occur; instead a ternary protein/template/d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features appear due to ring modes from the template/primer bases being perturbed and from the DNA backbone, as well as from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary than in the binary complex. Using standardized Raman intensities followed as a function of time C(*C)TP population in the crystal maximized at about 20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred.

Direct Evidence for a Peroxide Intermediate and a Reactive Enzyme–Substrate–Dioxygen Configuration in a Cofactor‐free Oxidase

“…; b) In crystallo Raman spectroscopy shows a dose‐dependent decrease of the 605 cm −1 5‐PMUA “fingerprint band” band; c) Decrease of Raman intensity is specific for the 605 cm −1 band. Two‐tailed p ‐value analysis indicates a significant dose correlation for this band (p‐value=0.0022), while the 622 cm −1 and 644 cm −1 bands assigned to Phe and Tyr, respectively,17 are unaffected. Local scaling was carried out using the 565 cm −1 band.…”

Large α‐helical movements observed in ternary crystals of RB69 DNA polymerase following nucleotide incorporation

“…We have seen similar bands in the RNA polymerase T. thermophilus crystals during translocation, and we have tentatively assigned them to strained noncanonical phosphodiester bonds present as the backbone makes a bend in the active site. In addition, bands in the 1000–1100 cm −1 region may have contributions from pyrophosphate . These assignments will be verified in the future by O, 13 C site‐specific labeling at a different backbone positions in the primer and template strands and in the triphosphate tail of dATP.…”

“…However, the absence of a second intense peak near 1245 cm −1 (another cytosine signature ring mode) rules out significant contribution from cytosine at 779 cm −1 . The second line of evidence is the high positive/negative changes observed in the 1000–1150 cm −1 triphosphate (~1125 cm −1 ) and pyrophosphate region . It was hypothesized that a negative triphosphate mode would appear at 1125 cm −1 ; however, this region of the spectrum is obscured because of the broad intensity near 1150 cm −1 , which may be attributed to a distorted region of the DNA backbone.…”

“…In addition, bands in the 1000-1100 cm À1 region may have contributions from pyrophosphate. [31] These assignments will be verified in the future by [18] O, 13 C sitespecific labeling at a different backbone positions in the primer and template strands and in the triphosphate tail of dATP.…”

“…The second line of evidence is the high positive/negative changes observed in the 1000-1150 cm À1 triphosphate (~1125 cm À1 ) and pyrophosphate region. [31] It was hypothesized that a negative triphosphate mode would appear at 1125 cm À1 ; however, this region of the spectrum is obscured because of the broad intensity near 1150 cm À1 , which may be attributed to a distorted region of the DNA backbone. Isotopic substitutions using 13 C and 18 O in the triphosphate group of dATP are needed to provide confirmation of band assignments.…”