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PEDS Advance Access originally published online on May 31, 2007
Protein Engineering Design and Selection 2007 20(6):267-271; doi:10.1093/protein/gzm019
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Using archaeal histones for precise DNA fragmentation

E. Azzoni1, D. Sblattero2, M. Licciulli1, R. Marzari1 and P. Edomi1,3

1 Department of Biology, University of Trieste, via Giorgieri 7, I-34127 Trieste, Italy 2 Department of Medical Sciences, University of Eastern Piedmont, via Solaroli 17, 28100 Novara, Italy

3 To whom correspondence should be addressed. E-mail: edomi{at}units.it


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
The fragmentation of DNA is a useful procedure for many molecular biology procedures. However, most methods used to fragment DNA are poorly controllable, and cannot be used to create small fragments. We describe a method to generate random DNA fragments of a predictable size to be cloned in expression vectors for the construction of display libraries. The DNA is allowed to form complexes with archaeal histones from Methanothermus fervidus (HMf) and the HMf/DNA core complex is naturally protected from nuclease DNaseI activity, giving rise to DNA fragments of ~60 bp and multiples thereof. We found that by varying the wt/wt ratio between DNA and HMf, the concentration of DNA and the incubation time with DNaseI, DNA fragments of desired size can be obtained. This approach should be applicable to the efficient fragmentation of DNA for the construction of phage display polypeptide libraries, as well as any other molecular biology procedures in which small DNA fragments of defined size are required.

Keywords: archaeal histones/DNA fragmentation/expression gene libraries/phage display


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
The burst of genome sequencing over the last decade has brought many interesting opportunities to researchers. Knowledge of the genome sequences of an organism is an essential prerequisite to understanding gene functions, and proteomic techniques such as expression libraries, provide the tools to analyze an entire complement of proteins. Both prokaryotic genomic DNA and eukaryotic cDNA are increasingly used to construct large display libraries using hosts which allow the expression of the cloned gene product on their surface as fusion proteins for the selection on binders of interest. In many cases, this process requires the shearing of DNA in fragments of a length suitable for expression as chimeric protein without disturbing the integrity of the host. In a recent paper (Zacchi et al., 2003Go), we described a novel expression vector which selects for open reading frames (ORFs) directly within a phage display context. This vector is designed to express short polypeptides (20 to 100 aa) on phage in order to provide a source of interactors for epitope mapping of antibodies or counterparts for every kind of protein–protein interactions and could, in principal, be extended to display random ORFs representing the coding potential of, or universe of epitopes expressed by, a whole organism. DNA fragmentation is a major issue in the construction of complex gene libraries in which the size and the distribution of the DNA sequences is essential to guarantee the quality of the library. Sonication or other mechanical shearing of DNA, often results in poor fragmentation accompanied by DNA damage, with an average DNA size greater than 300 bp. Enzymatic treatment with either restriction enzymes or endonucleases, such as DNaseI, is biased by recognition of specific sequences (Herrera and Chaires, 1994Go). In addition, the length of the fragments obtained by DNaseI digestion varies greatly according to the reaction conditions, including the amount of nuclease, the supplier or lot of the enzyme, the reaction temperature and the features of DNA substrates. As a consequence, treatment with DNases often results in non-reproducible outcomes or complete DNA degradation.

The archaeal histone from Methanothermus fervidus (HMf) is a DNA-binding protein that has been shown to form nucleosome-like structures typical of eukaryotic cells (Sandman et al., 1990Go). Although HMf binds to DNA molecules in vitro forming complexes that protect the DNA fragments from micrococcal nuclease digestion, the fragments produced (30, ~60 or multiple of ~60 bp in length) (Grayling et al., 1997Go) are smaller than those observed with eukaryotic nucleosomes. Here, we report a method to generate DNA sequences of ~60 bp or multiple to be cloned and expressed on phages, by using DNA protected by archaeal HMf.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains

The bacterial strain used in this study was DH5{alpha}F' (Gibco®, MD, US): F'/endA1 hsd17 (rK– mK+) supE44 thi-1 recA1 gyrA (Nalr) relA1 {Delta} (lacZYA-argF)U169 deoR (F80dlacD(lacZ)M15).

Plasmids

The phagemid pET28b-tTG (Novagen, Madison, WI, USA) is the expression vector in which the gene coding for human tissue transglutaminase (tTG) has been cloned and is 7370 bp long (Sblattero et al., 2000Go). The phagemid pPAO2 is a derivative of pDAN5 (Sblattero and Bradbury, 2000Go), specifically modified to select random DNA fragments on the basis of the cloning of in-frame ORFs (Zacchi et al., 2003Go).

Archaeal nucleosome assembly and digestion

The recombinant HMf purified protein was kindly provided by K. Sandman. The DNA fragmentation experiments were carried on with 1 µg of pET28-htTG, previously linearized with ScaI (NEB, MA, USA), incubated with increasing amounts of purified HMf ranging from a wt/wt ratio of 1:1 (HMf/DNA) to 10:1 in the presence of 50 mM Tris–HCl, 10 mM MnCl2, pH 7.5. The digestion was performed by adding 0.005 U of DNaseI (Sigma®, MO, USA) at 37°C for times ranging from 30 s to 10 min. The reaction was stopped adding 10 mM EDTA. For the subsequent procedures, the DNA was purified from the reaction mixture by phenol/chloroform extraction and resuspension in water. The scaling-up of DNA fragmentation process was investigated by using two different amount of DNA of 1 and 4 µg at a HMf/DNA ratio (wt/wt) of 4:1, incubated for extended times ranging from 2 to 150 min. For each incubation time, the average length of the DNA fragments was estimated by electrophoretic analysis (50 bp ladder was used, when a more accurate definition of bands size was necessary).

Library construction

The DNA fragments were purified by electrophoresis in a 2% agarose gel and recovered from the gel using the Qiaquick Gel Extraction kit (Qiagen, Hilden, Germany) and cloned in the pPAO2 phagemid vector as described (Zacchi et al., 2003Go). Briefly, the DNA fragments were blunt-ended by the addition of Pfu DNA polymerase, ligated to LIC linkers in a reaction mixture containing LIC Adaptors (20 µM), 1 x T4 DNA ligase buffer, T4 DNA ligase (NEB) overnight at 15°C. The fragments with ligated adaptors were PCR amplified using specific primers. To create the single stranded LIC tails in the plasmid, StuI-digested vector and adaptor-ligated inserts were treated with T4 DNA polymerase in the presence of dTTP (0.5 mM) and dATP (0.5 mM), respectively. Each microliter of ligation reaction was then used to transform 50 µl of electrocompetent DH5{alpha}F' cells.

Selection and sequencing

Sixty randomly picked colonies of transformed cells selected for resistance to ampicillin were analyzed for inserts by PCR with specific labeled primers and DNA sequencing with ABI 377 sequencer.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 Acknowledgements
 References
 
Experimental design

The study was undertaken to determine the experimental conditions to obtain DNA fragments of defined size by DNaseI digestion of purified DNA protected by HMf (Fig. 1). A series of experiments was carried out on plasmid pET28b-tTG DNA by varying the DNA/HMf ratio, the DNA quantity, the digestion time and the plasmid DNA characteristics, either linear or circular covalently closed (cccDNA). Other basic parameters (e.g. pH and ionic strength) of incubation reaction of HMf with DNA were those previously determined (Grayling et al., 1997Go). Following digestion, the DNA fragments were cloned in the phagemid vector pPAO2, which allows the selection of in-frame sequences and inspected for correspondence with true ORFs found within the pET28b-tTG plasmid.


Figure 1
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  		Fig. 1. The scheme for DNA fragmentation in presence of archaeal histone. HMf binds to DNA molecules forming complexes that protect DNA fragments from DNaseI digestion that are ~60 bp or multiple of ~60 bp in length.

 
HMf/DNA ratio and digestion time

DNaseI concentration was preliminary assayed by digesting pET28b-tTG with serial dilutions of the commercial enzyme. The working dilution was considered the highest dilution at which 1 µg of pET28b-tTG in 100 µl of reaction mixture was no more detectable in an agarose gel stained with ethidium bromide after 5 min. Fixed amounts of linearized pET28-tTG were preincubated with four concentrations of HMf at ratios HMf/DNA (wt/wt) of 1:1, 4:1, 6:1, 10:1 and allowed to form complexes for 10 min. DNaseI was then added for several incubation times. The results of the electrophoretic analysis of DNA fragmentation at 30 s and 10 min are reported in Fig. 2; additional incubations time (2 min) were reported for intermediate ratios (4:1 and 6:1). Fragments of ~60 bp in length (HMf resistant core) were considered the reference molecular weight to analyze the extent of protection from the DNaseI digestion. As previously reported (Grayling et al., 1997Go) at HMf/DNA ratios used (≥600 molar ratios) ~60 bp is the lowest length of the protected fragments. At a ratio wt/wt of 1:1, only a faint band of DNA of 60 bp was detectable after 30 s of digestion whereas at 10 min all the DNA was no more visible. At higher HMf concentration and longer incubation times, the DNA showed progressive electrophoretic shifts toward a more consistent low molecular weight band, a larger band and a smear of partly digested DNA. The better results were obtained with an incubation time of 10 min at a ratio wt/wt of 4:1 and 6:1, since at incubation time of 2 min, a smear was still observed and a ratio of 10:1 gave a majority of undigested DNA. Hence, the range of HMf concentration between 4:1 and 6:1 was considered useful to modulate the average size of the DNA fragments.


Figure 2
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  		Fig. 2. DNaseI digestion of HMf/DNA complexes. HMf /Plasmid pET28b-tTG DNA are formed at different wt/wt ratios (1 µg of plasmid) and exposed to DNaseI (0.005 U) for 30 s, 2 min or 10 min (each lane of the same ratio corresponds to a different sampling of the reaction); the untreated plasmid was loaded in the first lane. Plasmid DNA was ScaI linearized or not (c.c.c). Electrophoresis on 2% agarose gel (MW = 100 bp ladder).

 
Linear and circular covalently close DNA

To avoid the variability in shearing HMf/pET28b-tTG complex that may have been caused by a different degree in supercoiling of the cccDNA, a linearized plasmid was used in all the experiments. Anyway, a comparison between the level of protection of HMf on either linear DNA or cccDNA was performed. The results of the digestion of cccDNA plasmids at two times of incubation are reported in Fig. 2. Both linear and cccDNA showed the formation of one or more band in the lower part of the gel (compare 5th and 13th lanes of Fig. 2), although the fragmentation of circular plasmid showed a greater distribution of DNA lengths at the longer time (10 min) than the linear DNA.

Scaling-up of treated DNA

The effect of increasing quantities of DNA on the efficiency of the system was investigated using two amounts of 1 µg and 4 µg of pET28b-tTG digested for times ranging from 2 to 150 min, at a HMf/DNA ratio of 4:1. All samples were analyzed by electrophoresis registering the average size of the digested DNA and the corresponding values were used to calculate the regressions reported in Fig. 3. In the figure, the regression line intersects the x-axis at a value corresponding at a DNA fragment of 60 bp, which must be considered the DNA length resistant to further degradation. The x values at the intersections represent the times required to degrade a given amount of DNA to 60 bp.


Figure 3
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  		Fig. 3. Regression analysis of the average sizes of the DNA fragments of two amounts of DNA of 1 µg (triangles) and 4 µg (squares) after treatment of DNaseI (0.005 U) for increasing time. The HMf/DNA wt/wt ratio was 4:1. The x-axis is drawn at 60 bp to evidence that at prolonged time, all the DNA was converted to fragments around 60 bp.

 
PET tTG DNA fragment mapping

To determine the distribution of the fragments obtained from pET28b-tTG along the plasmid sequence, the fragmented DNA was cloned in the phagemid vector pPAO2. This phagemid was originally developed to make phage display polypeptides libraries and is based on the cloning of random DNA sequences upstream of a fusion gene, consisting of the ß-lactamase gene flanked by lox recombination sites, which is in turn upstream of gene 3 from fd phage. Only those clones containing DNA fragments encoding ORFs are able to confer ampicillin resistance, and so survive. PPAO2 requires the cloning of DNA sequences of predictable molecular weight to guarantee the correct expression on the surface on the phage of peptides with suitable length. In the present study, the plasmid DNA was first linearized with ScaI that cuts inside the tTG gene to analyze whether the free ends of the linear DNA were equally protected by HMf. Following cloning and plating of bacteria on ampicillin to select the in-frame sequences, 60 clones were randomly picked and sequenced. The individual sequences were compared and aligned to the plasmid sequence. The result is reported in Fig. 4. Almost all the fragments were shown to be different and covered the 63% of the plasmid length; having obtained 8899 bases of sequencing (i.e. 1.2 x coverage of the 7370 bp of the plasmid), this percentage is slightly lower than the 70% expected from a random cleavage. In addition, a couple of clones were found close to the 3' end of the plasmid, although none were found at the 5' end.


Figure 4
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  		Fig. 4. Sequence analysis of ampicillin-selected ORF pET28b-tTG fragments cloned in pPAO2. The plasmid pET28b-tTG is represented linearly and the individual clones (black bars of length proportional to their bp) located in correspondence of the homologous plasmid sequences. In the table, the number of clones that cover portions of the plasmid sequence without gaps and the correspondent percentage of the entire length are reported.

 
Distribution of fragments sizes

The lengths of the sequenced fragments were also analyzed to see whether the predicted dimension of 60 bp and multiples were still retained after digestion and cloning. All the fragment lengths were grouped according to intervals of 10 bp and the frequency of each class reported in Fig. 5. As can be seen, the lengths are distributed according to a multi-modal Gaussian curve with a mean value in the class of 140–150 bp and a modal class of 120–130 bp.


Figure 5
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  		Fig. 5. Frequency distribution of the 60 pETb-tTG fragments cloned in pPAO2, arranged according to the length in groups of 10 bp each; x labels are referred to average size of each class.

 

   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
Acknowledgements
 References
 
The method suggested in the present study has proved to be useful to overcome some of technical problems linked to the shearing of DNA for the construction of expression gene libraries in which small DNA fragments are required. The DNA-binding and nuclease-protection properties of the HMf histones were shown to significantly modify the activity of DNaseI, allowing the isolation of short DNA fragments. This is attested by the fact that even after prolonged digestions, at suitable HMf/DNA ratios (i.e. wt/wt 4:1 and 6:1), the DNA was never completely degraded. Only at a ratio wt/wt of 1:1, the DNA underwent complete degradation after 10 min, attesting a limited effect of protection. Interestingly, at a ratio of 10:1, the DNA was only partly fragmented and this opens the possibility of modulating the nuclease activity to obtain DNA fragments at a predetermined length under very controlled conditions. A first but more precise indication is provided by the experiments with different amounts of DNA and the regression lines calculated on the rates of degradation of the two DNAs. According to these relationships, it would be possible to calculate the time required to degrade a given amount of DNA to fragments of a specific length, once the source of DNase had been calibrated.

Other indications on the characteristics of the DNA fragmentation came from the small library constructed from the DNA of pET28b-tTG. This plasmid can be considered a minigenome containing the four plasmid genes accounting for ~50% of the DNA, other than the cloned tTG gene. Almost all the 60 clones sequenced were shown to be different to one another, without any over-representation of particular clones and a few clones overlapped one of the linearized DNA ends. The percentage of plasmid sequence covered with the clones sequencing is similar to the probability of sequence representation with a random cleavage, calculated considering 1.2 x coverage of the entire plasmid. So, we feel this method shows no bias for specific DNA sequences, even though DNaseI has proven to have specific recognition sequences (Herrera and Chaires, 1994Go). Regarding the preferential binding of archaeal histones to specific sequences (Bailey et al., 2000Go), it should be noted that these are different from the DNase recognized sites and were identified with several cycles of binding of which the first one was shown to be essentially random. To reduce the potential bias of DNase, the HMf/DNA ratio and the incubation time would be calibrated to achieve a partial digestion. Furthermore, we have preliminary evidences that with a modified recombinant version of histones, it is possible to clone single cDNA molecules and to obtain different phage clones covering all the coded protein (personal data).

With respect to the average size of the fragments, we have calculated a frequency distribution for the plasmid fragments cloned in pPAO2. This statistical analysis shown that the more frequent classes have a size corresponding to the predicted 60 bp and multiples protected by the histones, with a mean value in the range of 140–150 bp and a modal class in the range of 120–130 bp.

In conclusion, we have demonstrated the utility of this approach to shear DNA in fragments of approximately equal size with a lower limit of 60 bp, which cannot be obtained by any other means. Since the range of fragments to construct a library is usually chosen by the insert-size limitations, this method looks particularly favorable when specific expression vectors, such as phages are used.

Considering the above-mentioned personal data, we think that this system of fragmentation can be applied with different DNA molecules, and in particular could be useful for fragmenting cDNA and high gene density genomes.

Although we have developed this fragmentation approach for use in phage display and the identification of immunological epitopes, it is clear that it could also be used in any procedure in which small DNA fragments are required, such as the identification of DNA binding sites by transcription factors. This approach is likely to be far less biased than PCR based approaches (Singer et al., 1997Go), in which the interaction of nine random bases at the 3' end can be influenced by the sequence of the attached amplification primer.


   Footnotes
 
Edited by Andrew Bradbury


   Acknowledgements
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by FISM—Fondazione Italiana Sclerosi Multipla—Cod. 2004/R/6 to Edomi.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bailey K.A., Pereira S.L., Widom J., Reeve J.N. J. Mol. Biol. (2000) 303:25–34.[CrossRef][Web of Science][Medline]

Grayling R.A., Bailey K.A., Reeve J.N. Extremophiles (1997) 1:79–88.[CrossRef][Medline]

Herrera J.E., Chaires J.B. J. Mol. Biol. (1994) 236:405–411.[CrossRef][Web of Science][Medline]

Sandman K., Krzycki J.A., Dobrinski B., Lurz R., Reeve J.N. Proc. Natl Acad. Sci. USA (1990) 87:5788–5791.[Abstract/Free Full Text]

Sblattero D., Bradbury A. Nat. Biotechnol. (2000) 18:75–80.[CrossRef][Web of Science][Medline]

Sblattero D., Berti I., Trevisiol C., Marzari R., Tommasini A., Bradbury A., Fasano A., Ventura A., Not T. Am. J. Gastroenterol. (2000) 95:1253–1257.[CrossRef][Web of Science][Medline]

Singer B.S., Shtatlan T., Brown D., Gold L. Nucl. Acids Res. (1997) 25:781–786.[Abstract/Free Full Text]

Zacchi P., Sblattero D., Florian F., Marzari R., Bradbury A. Genome Res. (2003) 13:980–990.[Abstract/Free Full Text]

Received December 21, 2006; revised March 28, 2007; accepted April 12, 2007.


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This Article
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