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Timing facilitated site transfer of an enzyme on DNA.

ABSTRACT: Many enzymes that react with specific sites in DNA have the property of facilitated diffusion, in which the DNA chain is used as a conduit to accelerate site location. Despite the importance of such mechanisms in gene regulation and DNA repair, there have been few viable approaches to elucidate the microscopic process of facilitated diffusion. Here we describe a new method in which a small-molecule trap (uracil) is used to clock a DNA repair enzyme as it hops and slides between damaged sites in DNA. The 'molecular clock' provides unprecedented information: the mean length for DNA sliding, the one-dimensional diffusion constant, the maximum hopping radius and the time frame for DNA hopping events. In addition, the data establish that the DNA phosphate backbone is a sufficient requirement for DNA sliding.

SUBMITTER: Schonhoft JD

PROVIDER: S-EPMC3262087 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Timing facilitated site transfer of an enzyme on DNA.

Schonhoft Joseph D JD Stivers James T JT

Nature chemical biology 20120108 2

Many enzymes that react with specific sites in DNA have the property of facilitated diffusion, in which the DNA chain is used as a conduit to accelerate site location. Despite the importance of such mechanisms in gene regulation and DNA repair, there have been few viable approaches to elucidate the microscopic process of facilitated diffusion. Here we describe a new method in which a small-molecule trap (uracil) is used to clock a DNA repair enzyme as it hops and slides between damaged sites in ...[more]

PMID: 22231272

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Project description:Apolipoprotein B mRNA editing enzyme catalytic subunit 3 (APOBEC3) proteins are critical for the control of infection by retroviruses. These proteins deaminate cytidines in negative-strand DNA during reverse transcription, leading to G-to-A changes in coding strands. Uracil DNA glycosylase (UNG) is a host enzyme that excises uracils in genomic DNA, which the base excision repair machinery then repairs. Whether UNG removes uracils found in retroviral DNA after APOBEC3-mediated mutation is not clear, and whether this occurs in vivo has not been demonstrated. To determine if UNG plays a role in the repair of retroviral DNA, we used APOBEC3G (A3G) transgenic mice which we showed previously had extensive deamination of murine leukemia virus (MLV) proviruses. The A3G transgene was crossed onto an Ung and mouse Apobec3 knockout background (UNG-/-APO-/-), and the mice were infected with MLV. We found that virus infection levels were decreased in A3G UNG-/-APO-/- compared with A3G APO-/- mice. Deep sequencing of the proviruses showed that there were significantly higher levels of G-to-A mutations in proviral DNA from A3G transgenic UNG-/-APO-/- than A3G transgenic APO-/- mice, suggesting that UNG plays a role in the repair of uracil-containing proviruses. In in vitro studies, we found that cytoplasmic viral DNA deaminated by APOBEC3G was uracilated. In the absence of UNG, the uracil-containing proviruses integrated at higher levels into the genome than those made in the presence of UNG. Thus, UNG also functions in the nucleus prior to integration by nicking uracil-containing viral DNA, thereby blocking integration. These data show that UNG plays a critical role in the repair of the damage inflicted by APOBEC3 deamination of reverse-transcribed DNA. IMPORTANCE While APOBEC3-mediated mutation of retroviruses is well-established, what role the host base excision repair enzymes play in correcting these mutations is not clear. This question is especially difficult to address in vivo. Here, we use a transgenic mouse developed by our lab that expresses human APOBEC3G and also lacks the endogenous uracil DNA glycosylase (Ung) gene and show that UNG removes uracils introduced by this cytidine deaminase in MLV reverse transcripts, thereby reducing G-to-A mutations in proviruses. Furthermore, our data suggest that UNG removes uracils at two stages in infection-first, in unintegrated nuclear viral reverse-transcribed DNA, resulting in its degradation; and second, in integrated proviruses, resulting in their repair. These data suggest that retroviruses damaged by host cytidine deaminases take advantage of the host DNA repair system to overcome this damage.

Project description:In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1) (27.2 kJ mol(-1)) for the carbinolamine formation reaction and 44.5 kcal mol(-1) (186 kJ mol(-1)) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1) (62.8 kJ mol(-1)) in the forward (formation) reaction and 19 kcal mol(-1) (79.5 kJ mol(-1)) for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N-terminal amine.

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Timing facilitated site transfer of an enzyme on DNA.

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Timing facilitated site transfer of an enzyme on DNA.

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