Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzymedependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson−Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, highaccuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, ␣ and ␤, with proposed quaternary structures of ␣2␤2, ␣6␤2, or ␣6␤6, depending on the organism. The ␣ subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The ␤2 subunit houses the diferric Y • (1 radical per ␤2) cofactor that is required to initiate nucleotide reduction. 2 ,2 -Difluoro-2 -deoxycytidine (F2C) is presently used clinically in a variety of cancer treatments and the 5 -diphosphorylated F2C (F2CDP) is a potent inhibitor of RNRs. The studies with [1 -3 H]-F2CDP and [5-3 H]-F2CDP have established that F2CDP is a substoichiometric mechanism based inhibitor (0.5 eq F2CDP/␣) of both the Escherichia coli and the human RNRs in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 eq/␣) and is accompanied by release of 0.5 eq cytosine/␣. Inactivation also results in loss of 40% of ␤2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an ␣2␤2 tight complex is generated subsequent to enzyme inactivation by F2CDP, whereas in the human RNR, an ␣6␤6 tight complex is generated. Isolation of these complexes establishes that the weak interactions of the subunits in the absence of nucleotides are substantially increased in the presence of F2CDP and ATP. This information and the proposed asymmetry between the interactions of ␣n␤n provide an explanation for complete inactivation of RNR with substoichiometric amounts of F2CDP.G emcitabine, or 2Ј,2Ј-difluoro-2Ј-deoxycytidine (F 2 C), is a drug that is used clinically in the treatment of advanced pancreatic cancer and non-small cell lung carcinomas (1-3). In humans, F 2 C enters the cell via CNT-type or ENT-type transporters (4-6) and must be phosphorylated to exhibit its cytotoxicity. The monophosphate of F 2 C (F 2 CMP) is generated by deoxycytidine kinase (7) and is rapidly phosphorylated to the di-and triphosphates (F 2 CDP and F 2 CTP) (8, 9). Diphosphorylated F 2 C (F 2 CDP) is an irreversible inhibitor of ribonucleotide reductase (RNR) (10-13), and F 2 CTP functions as a chain terminator in the DNA polymerase reaction (14, 15). Differentially phosphorylated states of gemzar can also interfere with other enzymes involved in nucleotide metabolism. The mechanisms of cytotoxicity of F 2 C depend on the phosphorylated state of the inhibitor and are likely to be cell specific and multifactoral. Our recent synthesis of [1Ј-3 H]-F 2 CDP has provided the required tool to investigate the mechanism by which this molecule inactivates RNRs. Studies reported herein provide a previously unrecognized approach for RNR inhibition, one in which the mechanism based inhibitor (F 2 CDP) enhances the interactions between the two subunits of RNR preventing nucleotide reduction despite substoichiometric labeling.RNRs catalyze the conversion on nucleoside di(tri)phosphates to de...

DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows. Golden Gate assembly is a frequently employed DNA assembly methodology that utilizes a Type IIS restriction enzyme and a DNA ligase to generate recombinant DNA constructs from smaller DNA fragments. However, the utility of this methodology has been limited by a lack of resources to guide experimental design. For example, selection of the DNA sequences at fusion sites between fragments is based on broad assembly guidelines or pre-vetted sets of junctions, rather than being customized for a particular application or cloning project. To facilitate the design of robust assembly reactions, we developed a high-throughput DNA sequencing assay to examine reaction outcomes of Golden Gate assembly with T4 DNA ligase and the most commonly used Type IIS restriction enzymes that generate three-base and four-base overhangs. Next, we incorporated these findings into a suite of webtools that design assembly reactions using the experimental data. These webtools can be used to create customized assemblies from a target DNA sequence or a desired number of fragments. Lastly, we demonstrate how using these tools expands the limits of current assembly systems by carrying out one-pot assemblies of up to 35 DNA fragments. Full implementation of the tools developed here enables direct expansion of existing assembly standards for modular cloning systems (e.g. MoClo) as well as the formation of robust new high-fidelity standards.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside 5′-diphosphates to the corresponding deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits, α and β, for activity. Humans possess two β subunits: one involved in S phase DNA replication (β) and a second in mitochondrial DNA replication (β′ or p53R2) and potentially DNA repair. Gemcitabine (F 2 C) is used clinically as an anticancer agent and its phosphorylated metabolites target many enzymes involved in nucleotide metabolism, including RNR. The present investigation with α (specific activity of 400 nmol/min/mg) and β′ (0.6 Y•/β′2 and a specific activity of 420 nmol/min/mg) establishes F 2 CDP is a substoichiometric inactivator of RNR. Incubation of this α/β′ with [1′-3 H] F 2 CDP or [5-3 H] F 2 CDP and re-isolation of the protein by Sephadex G50 chromatography resulted in recovery 0.5 eq. of covalently bound sugar and 0.03 eq. of tightly associated cytosine to α2. SDS PAGE analysis (loaded without boiling) of the inactivated RNR, showed that 60% of α migrates as a 90 kDa protein and 40% as a 120 kDa protein. Incubation of [1′-3 H] F 2 CDP with active site mutants C444S/A, C218S/A, E431Q/D-α and the C-terminal tail C787S/A and C790S /A mutants, reveals that no sugar label is bound to the active site mutants of α and that in the case of C218S-α, α migrates as a 90 kDa protein. Analysis of the inactivated wt-α/β′ RNR by size exclusion chromatography indicates a quaternary structure of α6β′6. A mechanism of inactivation common with hα/β is presented.

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

Gemcitabine 5′-diphosphate (F 2 CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The E. coli RNR, an α2β2 complex, when incubated with one equivalent of F 2 CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of α with the sugar of F 2 CDP (one-label/α2β2). SDS PAGE analysis of the inactivated RNR without boiling of the sample reveals that α migrates as an 87 kDa and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-α in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-α migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR's active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1′-2 H] F 2 CDP was studied with wt-and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is nucleotide derived with g values of g x 2.00738, g y 2.00592, g z 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1′-1 H] F 2 CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR.

DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.

Background: T4 DNA ligase catalyzes the formation of phosphodiester bonds in dsDNA.Results: Single turnover rates of all chemical reaction steps exceed steady state turnover rates by 10-fold.Conclusion: Product release or a postligation conformational change is rate-limiting during turnover.Significance: This study represents the first detailed analysis of the kinetic mechanism of nick ligation by T4 DNA ligase.