Blunt-ended Substrates Research Articles

Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity.

Double-stranded DNA minicircles of less than 1000 bp in length have great interest in both fundamental research and therapeutic applications. Although minicircles have shown promising activity in gene therapy thanks to their good biostability and better intracellular trafficking, minicircles down to 250 bp in size have not yet been investigated from the test tube to the cell for lack of an efficient production method. Herein, we report a novel versatile plasmid-free method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide blunt-ended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2 μM. This one pot multi-step reaction based-method yields hundreds of micrograms of minicircle with sequences of any base composition and position and containing or not a variety of site-specifically chemical modifications or physiological supercoiling. Biochemical and cellular studies were then conducted to design a 95 bp minicircle capable of binding in vitro two NF-κB transcription factors per minicircle and to efficiently inhibiting NF-κB-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids.

Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

Engineered DNA ligases with improved activities in vitro

The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases.

Template‐independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1

Reverse transcriptases (RTs) possess a non-templated addition (NTA) activity while synthesizing DNA with blunt-ended DNA primer/templates. Interestingly, the RT of the long terminal repeat retrotransposon Tf1 has an NTA activity that is substantially higher than that of HIV-1 or murine leukemia virus RTs. By performing steady state kinetics, we found that the differences between the NTA activities of Tf1 and HIV-1 RTs can be explained by the substantially lower K(M) value for the incoming dNTP of Tf1 RT (while the differences between the apparent k(cat) values of these two RTs are relatively small). Furthermore, the K(M) values, calculated for both RTs with the same dNTP, are much lower for the template-dependent synthesis (TDS) than those of NTA. However, TDS of HIV-1 RT is higher than that of Tf1 RT. The overall relative order of the apparent k(cat)/K(M) values for dATP is: HIV-1 RT (TDS) > Tf1 RT (TDS) >> Tf1 RT (NTA) > HIV-1 RT (NTA). Under the employed conditions, Tf1 RT can add up to seven nucleotides to the blunt-ended substrate, while the other RTs add mostly a single nucleotide. The NTA activity of Tf1 RT is restricted to DNA primers. Furthermore, the NTA activity of Tf1 and HIV-1 RTs is suppressed by ATP, as it competes with the incoming dATP (although ATP is not incorporated by the NTA activity of the RTs). The unusually high NTA activity of Tf1 RT can explain why, after completing cDNA synthesis, the in vivo generated Tf1 cDNA has relatively long extra sequences beyond the highly conserved CA at its 3'-ends.

DNA Unwinding by ASCC3 Helicase Is Coupled to ALKBH3-Dependent DNA Alkylation Repair and Cancer Cell Proliferation

Demethylation by the AlkB dioxygenases represents an important mechanism for repair of N-alkylated nucleotides. However, little is known about their functions in mammalian cells. We report the purification of the ALKBH3 complex and demonstrate its association with the activating signal cointegrator complex (ASCC). ALKBH3 is overexpressed in various cancers, and both ALKBH3 and ASCC are important for alkylation damage resistance in these tumor cell lines. ASCC3, the largest subunit of ASCC, encodes a 3'-5' DNA helicase, whose activity is crucial for the generation of single-stranded DNA upon which ALKBH3 preferentially functions for dealkylation. In cell lines that are dependent on ALKBH3 and ASCC3 for alkylation damage resistance, loss of ALKBH3 or ASCC3 leads to increased 3-methylcytosine and reduced cell proliferation, which correlates with pH2A.X and 53BP1 foci formation. Our data provide a molecular mechanism by which ALKBH3 collaborates with ASCC to maintain genomic integrity in a cell-type specific manner.

The HIV-1 Integrase Monomer Induces a Specific Interaction with LTR DNA for Concerted Integration

The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO(4) was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3'-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19-42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.

Changes to the HIV Long Terminal Repeat and to HIV Integrase Differentially Impact HIV Integrase Assembly, Activity, and the Binding of Strand Transfer Inhibitors

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G(2)T(1)C(-1)A(-2)) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G(2):C(2) base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln(148) interacts with G(2), T(1), and C(-1) at the 5' end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.

Processing of Viral DNA Ends Channels the HIV-1 Integration Reaction to Concerted Integration*[boxs

Retroviral DNA made by reverse transcription is blunt-ended, and the viral integrase protein must remove two nucleotides from each 3′ end prior to integration into chromosomal DNA. Under most reaction conditions for integration in vitro, the majority of the reaction products are “half-site” products that result from integration of only one viral DNA end into one strand of the target DNA. Preprocessed DNA substrates are more efficient substrates for half-site reactions than are blunt-ended substrates, which require the removal of two nucleotides prior to integration. In contrast, we find that blunt-ended DNA is a better substrate for the biologically relevant reaction of concerted integration of pairs of viral DNA ends. The reaction pathway is channeled to concerted integration, and half-site integration products are reduced with blunt-ended DNA substrate that must first be processed by integrase. In addition, the terminal nucleotide requirements for concerted integration are more stringent than for the half-site reaction. Longer DNA is more efficient for the concerted reaction than is shorter DNA that is capable of efficient half-site integration. This suggests that nonspecific interactions of integrase with viral DNA distant from the termini contribute to the assembly of a complex that is competent for concerted integration. Finally, differential effects of mutation of a residue in the C-terminal domain of integrase on concerted versus half-site integration implicate protein-protein interactions involving this domain as important for concerted integration.

The Molecular Architecture of the Mammalian DNA Repair Enzyme, Polynucleotide Kinase

Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.

Substrate Requirements for Secondary Cleavage by HIV-1 Reverse Transcriptase RNase H

During and after minus-strand DNA synthesis, human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) degrades the RNA genome. To remove RNA left after polymerization, the RT aligns to cut 18 nucleotides in from the 5' RNA end. The enzyme then repositions, making a secondary cut 8 nucleotides from the RNA 5' end. Transfer of the minus strong stop DNA during viral replication requires cleavage of template RNA. Removal of the terminal RNA segment is a special case because the RNA-DNA hybrid forms a blunt end, shown previously to resist cleavage when tested in vitro. We show here that the structure of the substrate extending beyond the RNA 5' end is an important determinant of cleavage efficiency. A short single-stranded DNA extension greatly stimulated the secondary cleavage. Annealing an RNA segment to the DNA extension was even more stimulatory. Recessing the DNA from a blunt end by even one nucleotide caused the RT to reorient its binding, preventing secondary cleavage. The presence of the cap at the 5' end of the viral RNA did not improve the efficiency of secondary cleavage. However, NC protein greatly facilitated the secondary cut on the blunt-ended substrate, suggesting that NC compensates for the unfavorable substrate structure.

Eukaryotic ribonucleases HI and HII generate characteristic hydrolytic patterns on DNA-RNA hybrids: further evidence that mitochondrial RNase H is an RNase HII.

RNase H activities from HeLa cells (either of cytoplasmic or mitochondrial origin), and from mitochondria of beef heart and Xenopus ovaries, have been tested with RNA-DNA substrates of defined length (20 bp) and sequence. Substrates were either blunt-ended, or presented DNA or RNA overhangs. The hydrolysis profiles obtained at early times of the digestion showed a good correlation between the class of RNase H, either type I or II assigned according to biochemical parameters, whatever the organism. Consequently, the pattern of primary cuts can be considered as a signature of the predominant RNase H activity. For a given sequence, hydrolysis profiles obtained are similar, if not identical, for either blunt-ended substrates or those presenting overhangs. However, profiles showed variations depending on the sequence used. Of the three sequences tested, one appears very discriminatory, class I RNases H generating a unique primary cut 3 nt from the 3' end of the RNA strand, whereas class II RNases H generated two simultaneous primary cuts at 6 and at 8 nt from the 5' end of the RNA strand. Hydrolysis profiles further confirm the assignation of the mitochondrial RNase H activity from HeLa cells, beef heart and Xenopus oocytes to the class II.