One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse genetics system.

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design.

Large DNA constructs (>10 kb) are invaluable tools for genetic engineering and the development of therapeutics. However, the manufacture of these constructs is laborious, often involving multiple hierarchical rounds of preparation. To address this problem, we sought to test whether Golden Gate assembly (GGA), an in vitro DNA assembly methodology, can be utilized to construct a large DNA target from many tractable pieces in a single reaction. While GGA is routinely used to generate constructs from 5 to 10 DNA parts in one step, we found that optimization permitted the assembly of >50 DNA fragments in a single round. We applied these insights to genome construction, successfully assembling the 40 kb T7 bacteriophage genome from up to 52 parts and recovering infectious phage particles after cellular transformation. The assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Mismatch discrimination and sequence bias during end-joining by DNA ligases.

DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3'-OH and 5'-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.

Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design.

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.

Structural snapshots of human DNA polymerase µ engaged on a DNA double-strand break.

Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase µ (Polµ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polµ engaging a DSB substrate. Synapsis is mediated solely by Polµ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol µ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polµ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.

Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent 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, high-accuracy 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 incorporation enables repair of chromosome breaks by nonhomologous end joining.

The nonhomologous end-joining (NHEJ) pathway preserves genome stability by ligating the ends of broken chromosomes together. It employs end-processing enzymes, including polymerases, to prepare ends for ligation. We show that two such polymerases incorporate primarily ribonucleotides during NHEJ-an exception to the central dogma of molecular biology-both during repair of chromosome breaks made by Cas9 and during V(D)J recombination. Moreover, additions of ribonucleotides but not deoxynucleotides effectively promote ligation. Repair kinetics suggest that ribonucleotide-dependent first-strand ligation is followed by complementary strand repair with deoxynucleotides, then by replacement of ribonucleotides embedded in the first strand with deoxynucleotides. Our results indicate that as much as 65% of cellular NHEJ products have transiently embedded ribonucleotides, which promote flexibility in repair at the cost of more fragile intermediates.

Polµ tumor variants decrease the efficiency and accuracy of NHEJ.

The non homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair often requires DNA synthesis to fill the gaps generated upon alignment of the broken ends, a complex task performed in human cells by two specialized DNA polymerases, Polλ and Polµ. It is now well established that Polµ is the one adapted to repair DSBs with non-complementary ends, the most challenging scenario, although the structural basis and physiological implications of this adaptation are not fully understood. Here, we demonstrate that two human Polµ point mutations, G174S and R175H, previously identified in two different tumor samples and affecting two adjacent residues, limit the efficiency of accurate NHEJ by Polµ in vitro and in vivo. Moreover, we show that this limitation is the consequence of a decreased template dependency during NHEJ, which renders the error-rate of the mutants higher due to the ability of Polµ to randomly incorporate nucleotides at DSBs. These results highlight the relevance of the 8 kDa domain of Polµ for accurate and efficient NHEJ, but also its contribution to the error-prone behavior of Polµ at 2-nt gaps. This work provides the first demonstration that mutations affecting Polµ identified in tumors can alter the efficiency and fidelity of NHEJ.

Structural accommodation of ribonucleotide incorporation by the DNA repair enzyme polymerase Mu.

While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase µ (Pol µ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol µ with a ribonucleotide bound at the active site. These structures reveal that Pol µ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol µ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol µ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.

Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining.

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.

Essential role for polymerase specialization in cellular nonhomologous end joining.

Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) µ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol µ using the unpaired base adjacent to the downstream 5' phosphate even when there are available template sites further upstream of this position; this makes Pol µ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol µ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.

The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining.

Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures; however, it is unclear how the steps employed for resolution are determined. We sought to address this question by analysing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85 to 6%. End-processing by nucleases and polymerases is increased to compensate, although paths with the fewest number of steps to generate a substrate suitable for ligation are favoured. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors.

Nonhomologous end joining: a good solution for bad ends.

Double strand breaks pose unique problems for DNA repair, especially when broken ends possess complex structures that interfere with standard DNA transactions. Nonhomologous end joining can use multiple strategies to solve these problems. It further uses sophisticated means to ensure the strategy chosen provides the ideal balance of flexibility and accuracy.

Sustained active site rigidity during synthesis by human DNA polymerase µ.

DNA polymerase µ (Pol µ) is the only template-dependent human DNA polymerase capable of repairing double-strand DNA breaks (DSBs) with unpaired 3' ends in nonhomologous end joining (NHEJ). To probe this function, we structurally characterized Pol µ's catalytic cycle for single-nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, Pol µ shows no large-scale conformational changes in protein subdomains, amino acid side chains or DNA upon dNTP binding or catalysis. Instead, the only major conformational change is seen earlier in the catalytic cycle, when the flexible loop 1 region repositions upon DNA binding. Pol µ variants with changes in loop 1 have altered catalytic properties and are partially defective in NHEJ. The results indicate that specific loop 1 residues contribute to Pol µ's unique ability to catalyze template-dependent NHEJ of DSBs with unpaired 3' ends.

Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol.

Previous studies showed that fitter yeast (Saccharomyces cerevisiae) that can grow by fermenting glucose in the presence of allyl alcohol, which is oxidized by alcohol dehydrogenase I (ADH1) to toxic acrolein, had mutations in the ADH1 gene that led to decreased ADH activity. These yeast may grow more slowly due to slower reduction of acetaldehyde and a higher NADH/NAD(+) ratio, which should decrease the oxidation of allyl alcohol. We determined steady-state kinetic constants for three yeast ADHs with new site-directed substitutions and examined the correlation between catalytic efficiency and growth on selective media of yeast expressing six different ADHs. The H15R substitution (a test for electrostatic effects) is on the surface of ADH and has small effects on the kinetics. The H44R substitution (affecting interactions with the coenzyme pyrophosphate) was previously shown to decrease affinity for coenzymes 2-4-fold and turnover numbers (V/Et) by 4-6-fold. The W82R substitution is distant from the active site, but decreases turnover numbers by 5-6-fold, perhaps by effects on protein dynamics. The E67Q substitution near the catalytic zinc was shown previously to increase the Michaelis constant for acetaldehyde and to decrease turnover for ethanol oxidation. The W54R substitution, in the substrate binding site, increases kinetic constants (Ks, by >10-fold) while decreasing turnover numbers by 2-7-fold. Growth of yeast expressing the different ADHs on YPD plates (yeast extract, peptone and dextrose) plus antimycin to require fermentation, was positively correlated with the log of catalytic efficiency for the sequential bi reaction (V1/KiaKb=KeqV2/KpKiq, varying over 4 orders of magnitude, adjusted for different levels of ADH expression) in the order: WT≈H15R>H44R>W82R>E67Q>W54R. Growth on YPD plus 10mM allyl alcohol was inversely correlated with catalytic efficiency. The fitter yeast are "bradytrophs" (slow growing) because the ADHs have decreased catalytic efficiency.

Structure and functional analysis of the BRCT domain of translesion synthesis DNA polymerase Rev1.

Translesion synthesis (TLS) is a pathway in which specialized, low-fidelity DNA polymerases are used to overcome replication blocks caused by DNA damage. The use of this pathway often results in somatic mutations that can drive carcinogenesis. Rev1 is a TLS polymerase found in all eukaryotes that plays a pivotal role in mediating DNA damage-induced mutagenesis. It possesses a BRCA1 C-terminal (BRCT) domain that is required for its function. The rev1-1 allele encodes a mutant form of Rev1 with a G193R substitution in this domain, which reduces the level of DNA damage-induced mutagenesis. Despite its clear importance in mutagenic TLS, the role of the BRCT domain is unknown. Here, we report the X-ray crystal structure of the yeast Rev1 BRCT domain and show that substitutions in residues constituting its phosphate-binding pocket do not affect mutagenic TLS. This suggests that the role of the Rev1 BRCT domain is not to recognize phosphate groups on protein binding partners or on DNA. We also found that residue G193 is located in a conserved turn region of the BRCT domain, and our in vivo and in vitro studies suggest that the G193R substitution may disrupt Rev1 function by destabilizing the fold of the BRCT domain.

Pre-steady state kinetic studies show that an abasic site is a cognate lesion for the yeast Rev1 protein.

Rev1 is a eukaryotic DNA polymerase that rescues replication forks stalled at sites of DNA damage by inserting nucleotides opposite the damaged template bases. Yeast genetic studies suggest that Rev1 plays an important role in rescuing replication forks stalled at one of the most common forms of DNA damage, an abasic site; however, steady state kinetic studies suggest that an abasic site acts as a significant block to nucleotide incorporation by Rev1. Here we examined the pre-steady state kinetics of nucleotide incorporation by yeast Rev1 with damaged and non-damaged DNA substrates. We found that yeast Rev1 is capable of rapid nucleotide incorporation, but only a small fraction of the protein molecules possessed this robust activity. We characterized the nucleotide incorporation by the catalytically robust fraction of yeast Rev1 and found that it efficiently incorporated dCTP opposite a template abasic site under pre-steady state conditions. We conclude from these studies that the abasic site is a cognate lesion for Rev1.

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases.

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t(50)(bypass)) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t(50)(bypass) values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.

Variations on a theme: eukaryotic Y-family DNA polymerases.

Most classical DNA polymerases, which function in normal DNA replication and repair, are unable to synthesize DNA opposite damage in the template strand. Thus in order to replicate through sites of DNA damage, cells are equipped with a variety of nonclassical DNA polymerases. These nonclassical polymerases differ from their classical counterparts in at least two important respects. First, nonclassical polymerases are able to efficiently incorporate nucleotides opposite DNA lesions while classical polymerases are generally not. Second, nonclassical polymerases synthesize DNA with a substantially lower fidelity than do classical polymerases. Many nonclassical polymerases are members of the Y-family of DNA polymerases, and this article focuses on the mechanisms of the four eukaryotic members of this family: polymerase eta, polymerase kappa, polymerase iota, and the Rev1 protein. We discuss the mechanisms of these enzymes at the kinetic and structural levels with a particular emphasis on how they accommodate damaged DNA substrates. Work over the last decade has shown that the mechanisms of these nonclassical polymerases are fascinating variations of the mechanism of the classical polymerases. The mechanisms of polymerases eta and kappa represent rather minor variations, while the mechanisms of polymerase iota and the Rev1 protein represent rather major variations. These minor and major variations all accomplish the same goal: they allow the nonclassical polymerases to circumvent the problems posed by the template DNA lesion.

An alternative form of replication protein a prevents viral replication in vitro.

Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The "canonical" RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting "alternative" RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the canonical RPA complex; however, aRPA is unable to support DNA replication and inhibits canonical RPA function. Two regions of RPA4, the putative L34 loop and the C terminus, are responsible for inhibiting SV40 DNA replication. Given that aRPA inhibits canonical RPA function in vitro and is found in nonproliferative tissues, these studies indicate that RPA4 expression may prevent cellular proliferation via replication inhibition while playing a role in maintaining the viability of quiescent cells.