Cohesive DNA Ends Research Articles

Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning.

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories.

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway.

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

High G/C content of cohesive overhangs renders DNA end joining Ku-independent

Ku plays an important role in the repair of double strand DNA breaks by non-homologous DNA end joining (NHEJ). Ku is thought to exert its function by aligning the two DNA ends. A previous study showed that the joining of certain cohesive DNA ends in cell-free in vitro reactions was independent of Ku [Mol. Cell. Biol. 19 (1999) 2585]. To investigate a possible correlation between Ku-dependence of DNA end joining reactions and the strength of base pair interactions between cohesive ends, we constructed a series of repair substrates with either 3′- or 5′-overhangs, which consisted entirely of either A/T or G/C residues. We found that after Ku-immunodepletion of the extract, the joining of cohesive ends that associate by the formation of four A:T base pairs was reduced, while the joining of ends that associate through four G:C base pairs was unaffected or slightly stimulated. The precision of the repair was not reduced in Ku-independent reactions. Our results indicate that the requirement for Ku is dependent on how stably the two cohesive DNA ends can associate by base-pairing. Two independent assays for protein–DNA interactions did not reveal any differences in Ku binding to substrates with A/T and G/C overhangs, suggesting that in this system Ku is recruited to the repair site regardless of whether it is functionally required or not. The finding that Ku is dispensable for efficient and precise joining of ends with cohesive G/C overhangs also suggests that alignment of DNA ends may be the sole function of Ku during NHEJ.

Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction.

The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells. Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay. To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair. We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts. Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence. Factor activity was inhibited by anti-Mre11 antibody. The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified. Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation.

Increased cloning efficiency by temperature-cycle ligation.

Molecular cloning is a crucial, and often speed-limiting, step in many standard procedures of molecular biology. Ligation of cohesive DNA ends is normally carried out at 12–16 C to ensure a good balance between enzyme activity and stability of annealed DNA overhangs. Low temperatures generally reduce ligase activity, whereas too high temperatures may reduce cloning efficiencies by melting annealed DNA overhangs and increase overall molecular motion in the ligation reaction. Several procedures have been described to increase the efficiency ligation reactions, including the addition of condensing agents as polyethylene glycol (1) or hexamminedicobalt chloride (2). These approaches induce macromolecular crowding, and thus serve to mimic higher DNA concentrations in the ligation reaction. Other approaches seek to increase the efficiency of molecular cloning procedures by omitting the ligation step by generating long single-stranded DNA overhangs that can be annealed and transformed directly into an appropriate Escherichia coli host (3). Ligation of blunt-ended DNA fragments is normally carried out at room temperature using higher concentrations of T4 DNA ligase. We have devised a simple procedure in which high enzyme activity and DNA annealing is balanced by constant temperature cycling. We find temperature-cycle ligations (TCL) may increase the efficiency of staggered cut cloning ∼4–8-fold, while the efficiency of blunt-end clonings are increased ∼4–6-fold (see Table 1). The bacterial cloning vector pBluescript II KS+ was digested with AflIII and HindIII producing two cohesive end-fragments of 434 and 2526 bp respectively, or PvuII generating two blunt-end fragments of 448 and 2512 bp respectively. All enzymes were purchased from Amersham. Digested plasmid DNA was separated on 1% low-melting agarose (NuSieve) and purified as described by Sambrook et al. (1). For each digestion a ligation master mix was prepared containing 50 mM Tris–HCl (pH 7.6), 5 mM MgCl2, 5 mM DTT, 50 μg BSA/ml, 0.5 mM ATP, 200 ng DNA and 0.1 Weiss unit T4 DNA ligase/10 μl. The purified fragments were added in equimolar amounts. The master mix was divided into 10 μl ligation reactions and either incubated at 14 C, at room temperature (22 C), or subjected to temperature cycling. Temperature-cycle ligations were carried out for 12–16 h in a Perkin-Elmer-Cetus DNA Thermal Cycler 480 programmed indefinitely to cycle between 30 s at 10 C and 30 s at 30 C. In our setting, the machine performed 10 cycles/h. Competent XL1-Blue E.coli cells were transformed by electrotransformation according to manufacturer’s directions (BioRad), transferred to 1 ml LB medium and incubated at 37 C for 1 h. For enumeration of cloning efficiencies aliquots of 25–100 μl were spread onto LB plates containing 50 μg/ml ampicillin. The number of colonies appearing per transformation is shown in Table 1. We have tested the kinetics of temperature-cycle ligations over 12–16 h, and found them to be similar to those of ligations at constant temperatures resulting in a linear increase with time in the number of colonies appearing after transformation (data not shown). Furthermore, temperature-cycle ligation may be used together with hexamminedicobalt chloride in blunt-end ligations to give the combined effect.

The cos region of lactococcal bacteriophage phi LC3.

The nucleotide sequence of the DNA regions flanking the cohesive end site of the temperate lactococcal bacteriophage phi LC3 was determined. Four pairs of sequence repeats of 8 to 13 base-pairs in length were revealed close to the cohesive ends. These repeats possibly represent signal sequences involved in phage DNA maturation and packaging. The start of an open reading frame was localized only 42 nucleotides from the left end of the phi LC3 phage double-stranded region, implying that transcription probably proceeds across the cos site. Compilation of the characteristics of the DNA termini of bacteriophages with complementary cohesive DNA ends suggests that phages of Gram-negative bacteria generally carry 5'-protruding single-strands whereas those of Gram-positive hosts carry 3'-protruding ends.

Specific and reversible inhibition of the blunt end joining activity of the T4 DNA ligase.

Specific, complete and reversible inhibition of the joining of blunt ended DNA duplexes catalyzed by the T4 DNA ligase can be obtained by using ATP, the enzyme cofactor, at concentrations of 5 mM and higher. On cohesive DNA ends, 5 mM ATP, which completely inhibits blunt end ligation, brings about only a limited (8%) reduction in the level of joining obtainable under optimal ATP concentration (0,5 mM or lower). A similar but less drastic uncoupling of the two kinds of joining can be achieved at lower ATP concentration (2,5 mM) using 1 mM Mg++. The joining of DNA blunt ends can also be inhibited almost completely by 10 mM spermidine, without noticeable effect on the joining of cohesive termini.