Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors.

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for 'capture' by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification.

Efficient engineering of human and mouse primary cells using peptide-assisted genome editing.

Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.

Action and timing of BacC and BacD in the late stages of biosynthesis of the dipeptide antibiotic bacilysin.

Biosynthesis of the dipeptide antibiotic bacilysin, encoded by the seven Bacillus subtilis genes bacA-G, involves diversion of flux from prephenate to the noncognate amino acid anticapsin. The anticapsin warhead is then ligated to the C-terminus of l-alanine to produce mature bacilysin. We have previously noted the formation of two diastereomers of tetrahydrotyrosine (4S- and 4R-H(4)Tyr) by tandem action of the four purified enzymes BacABGF. BacC (oxidase) and BacD (ligase) have been hypothesized to be remaining late stage enzymes in bacilysin biosynthesis. Using a combination of BacCD in vitro studies, B. subtilis deletion mutants, and isotopic feeding studies, we were able to determine that the H(4)Tyr diastereomers are actually shunt products that are not on-pathway to bacilysin biosynthesis. Dihydroanticapsin and dihydrobacilysin accumulate in extracts of a ΔbacC strain and are processed to anticapsin and then bacilysin upon addition of BacC and BacD, respectively. These results suggest the epoxide group in bacilysin is installed in an earlier step of bacilysin biosynthesis, while BacC oxidation of the C(7)-hydroxyl and the subsequent BacD ligation of anticapsin to l-Ala are the penultimate and ultimate steps of bacilysin biosynthesis, respectively.

Stereochemical outcome at four stereogenic centers during conversion of prephenate to tetrahydrotyrosine by BacABGF in the bacilysin pathway.

The first four enzymes of the bacilysin antibiotic pathway, BacABGF, convert prephenate to a tetrahydrotyrosine (H(4)Tyr) diastereomer on the way to the anticapsin warhead of the dipeptide antibiotic. BacB takes the BacA product endocyclic-Δ(4),Δ(8)-7R-dihydrohydroxyphenylpyruvate (en-H(2)HPP) and generates a mixture of 3E- and 3Z-olefins of the exocyclic-Δ(3),Δ(5)-dihydrohydroxyphenylpyruvate (ex-H(2)HPP). The NADH-utilizing BacG then catalyzes a conjugate reduction, adding a pro-S hydride equivalent to C(4) to yield tetrahydrohydroxyphenylpyruvate (H(4)HPP), a transamination away (via BacF) from 2S-H(4)Tyr. Incubations of the pathway enzymes in D(2)O yield deuterium incorporation at C(8) from BacA and then C(9) from BacB action. By (1)H NMR analysis of samples of H(4)Tyr, the stereochemistry at C(4), C(8), and C(9) can be assigned. BacG (followed by BacF) converts 3E-ex-H(2)HPP to 2S,4R,7R-H(4)Tyr. The 3Z isomer is instead reduced and transaminated to the opposite diastereomer at C(4), 2S,4S,7R-H(4)Tyr. Given that bacilysin has the 2S,4S stereochemistry in its anticapsin moiety, it is likely that the 2S,4S-H(4)Tyr is the diastereomer "on pathway". NMR determination of the stereochemistry of the CHD samples at C(8) and C(9) allows assignment of all stereogenic centers (except C(3)) in this unusual tetrahydro-aromatic amino acid building block, giving insights into and constraints on the BacA, BacB, and BacG mechanisms.

Olefin isomerization regiochemistries during tandem action of BacA and BacB on prephenate in bacilysin biosynthesis.

BacA and BacB, the first two enzymes of the bacilysin pathway, convert prephenate to an exocylic regioisomer of dihydrohydroxyphenylpyruvate (ex-H(2)HPP) on the way to the epoxycyclohexanone warhead in the dipeptide antibiotic, bacilysin. BacA decarboxylates prephenate without aromatization, converting the 1,4-diene in prephenate to the endocyclic 1,3-diene in Δ(4),Δ(8)-dihydrohydroxyphenylpyruvate (en-H(2)HPP). BacB then performs an allylic isomerization to bring the diene into conjugation with the 2-ketone in the product Δ(3),Δ(5)-dihydrohydroxyphenylpyruvate (ex-H(2)HPP). To prove that BacA acts regiospecifically on one of the two prochiral olefins in prephenate, we generated 1,5,8-[(13)C]-chorismate from bacterial fermentation of 5-[(13)C]-glucose and in turn produced 2,4,6-[(13)C]-prephenate via chorismate mutase. Tandem action of BacA and BacB gave 2,4,8-[(13)C]-7R-ex-H(2)HPP, showing that BacA isomerizes only the pro-R double bond in prephenate. Nonenzymatic isomerization of the BacA product into conjugation gives only the Δ(3)E-geometric isomer of Δ(3),Δ(5)-ex-H(2)HPP. On the other hand, acceleration of the allylic isomerization by BacB gives a mixture of the E- and Z-geometric isomers of the 7R- product, indicating some rerouting of the flux, likely through dienolate geometric isomers.

Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA.

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1, and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP and dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth occurs in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U·A base pairs derived from dUTP incorporation, an isogenic ung(-/-) DT40 cell line showed little change in its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A or G or present in single-stranded DNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, the nature of the resulting DNA lesion, and the DNA repair response are discussed.

Dynamics of uracil and 5-fluorouracil in DNA.

The prodrug 5-fluorouracil (5-FU), after activation into 5-F-dUMP, is an extensively used anticancer agent that inhibits thymidylate synthase and leads to increases in dUTP and 5-F-dUTP levels in cells. One mechanism for 5-FU action involves DNA polymerase mediated incorporation of dUTP and 5-F-dUTP into genomic DNA leading to U/A, 5-FU/A, or 5-FU/G base pairs. These uracil-containing lesions are recognized and excised by several human uracil excision repair glycosylases (hUNG2, hSMUG2, and hTDG) leading to toxic abasic sites in DNA that may precipitate cell death. Each of these enzymes uses an extrahelical base recognition mechanism, and previous studies with UNG have shown that extrahelical recognition is facilitated by destabilized base pairs possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of base pairs containing 5-FU and U are an important unknown in understanding the role of these enzymes in damage recognition and prodrug activation. The pH dependence of the (19)F NMR chemical shift of 5-FU imbedded in a model trinucleotide was used to obtain a pK(a) = 8.1 for its imino proton (10 °C). This is about 1.5 units lower than the imino protons of uracil or thymine and indicates that at neutral pH 5-FU exists significantly as an ionized tautomer that can mispair with guanine during DNA replication. NMR imino proton exchange measurements show that U/A and 5-FU/A base pairs open with rate constants (k(op)) that are 6- and 13-fold faster than a T/A base pair in the same sequence context. In contrast, these same base pairs have apparent opening equilibrium constants (αK(op)) that differ by less than a factor of 2, indicating that the closing rates (k(cl)) are enhanced by nearly equal amounts as k(op). These dynamic measurements are consistent with the previously proposed kinetic trapping model for extrahelical recognition by UNG. In this model, the enhanced intrinsic opening rates of destabilized base pairs allow the bound glycosylase to sample dynamic extrahelical excursions of thymidine and uracil bases as the first step in recognition.

Impact of linker strain and flexibility in the design of a fragment-based inhibitor.

The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to high-affinity inhibitors. Ideally, this strategy would use linkers that do not perturb the optimal binding geometries of the fragments and do not have excessive conformational flexibility that would increase the entropic penalty of binding. In reality, these aims are seldom realized owing to limitations in linker chemistry. Here we systematically explore the energetic and structural effects of rigid and flexible linkers on the binding of a fragment-based inhibitor of human uracil DNA glycosylase. Analysis of the free energies of binding in combination with cocrystal structures shows that the flexibility and strain of a given linker can have a substantial impact on binding affinity even when the binding fragments are optimally positioned. Such effects are not apparent from inspection of structures and underscore the importance of linker optimization in fragment-based drug discovery efforts.

Uracil DNA glycosylase: revisiting substrate-assisted catalysis by DNA phosphate anions.

Uracil DNA glycosylase (UNG) is a powerful DNA repair enzyme that has been shown to stabilize a glycosyl cation reaction intermediate and a related tight binding inhibitor using electrostatic interactions with the +1 and -1, but not the +2, phosphodiester group of the single-stranded DNA substrate Ap (2+)Ap (1+)Up (1-)ApA. These experimental results differed considerably from computational findings using duplex DNA, where the +2 phosphate was found to stabilize the transition state by approximately 5 kcal/mol, suggesting that UNG uses different catalytic strategies with single-stranded and double-stranded DNA substrates. In addition, the computational studies indicated that the conserved and positively charged His148 (which hydrogen bonds to the +2 phosphate) destabilized the glycosyl cation intermediate by 6-8 kcal/mol through anticatalytic electrostatic interactions. To evaluate these interesting proposals, we measured the kinetic effects of neutral methylphosphonate (MeP) stereoisomers at the +1 and +2 positions of a 12-mer dsDNA substrate and also the catalytic contribution and ionization state of His148. For MeP substitutions at the +1 position, single-turnover kinetic studies showed that the activation barrier was increased by 9.8 and 3.1 kcal/mol, corresponding to a stereoselectivity of nearly 40000-fold for the respective MeP isomers. Identical to the findings with ssDNA, MeP substitutions at the +2 position resulted in only small changes in the activation barrier (+/-0.3 kcal/mol), with little stereoselectivity ( approximately 4-fold). However, the H148A mutation destabilizes both the ground state and transition states by 2.4 and 4.3 kcal/mol, respectively. Thus, His148 is catalytic because it stabilizes the transition state to a greater extent (1.9 kcal/mol) than the ground state. Heteronuclear NMR studies established that His148 was neutral in the free enzyme at neutral pH, and in conformational exchange in a specific DNA complex containing uracil. We conclude that the +1 and +2 phosphate esters play identical catalytic roles in the reactions of single-stranded and double-stranded DNA substrates, and that His148 serves a catalytic role by positioning the substrate and catalytic water, or by an environmental effect.

Enzymatic capture of an extrahelical thymine in the search for uracil in DNA.

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.