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370 SKOWRONEK AND BUJNICKI

enzymes recognizing novel sequences. Despite the fact that over 3000 REases

with over 250 different sequence specificities have been isolated from various

bacterial strains, many possible specificities have not yet been discovered (see

e.g. http://rebase.neb.com/cgi-bin/classlist). Unfortunately, the success rate for the

discovery of novel REases has decreased to a few enzymes per thousands of

screened strains over the last several years. The number of experimentally charac-

terized HEases is even smaller, currently less than 200 (however, unlike REases,

members of this group of nucleases usually display unique specificities). Thus,

there have been substantial efforts to engineer the known REases and HEases to

endow them with novel properties, by random mutagenesis as well as rational, i.e.

structure-guided, design.

Structural data from solved co-crystals of REeases with their cognate DNA

sequences indicate that these enzymes employ redundant protein-DNA contacts

involving multiple hydrogen bonds to the target sequence, also via water molecules,

and combine direct (sequence-based) and indirect (structure-based) read-out for

DNA recognition. This saturation and redundancy of protein-DNA contacts allows

extraordinary sequence specificity, but also has to be overcome to perform

successful protein engineering. REases also often couple recognition with catalysis.

Therefore, substitutions of residues involved in base-specific contacts typically lead

to null mutants, incapable of either DNA binding or cleavage. Thus far, most

attempts to modify the specificity of REases by rational mutagenesis of residues

found to be involved in protein-DNA interactions remained unsuccessful (reviews:

(Alves and Vennekohl, 2004; Lanio et al., 2000)) and only recently some variants

with altered specificities have been created by random mutagenesis and screening

or selection of mutants. On the other hand, crystal structures of HEases bound to

the DNA revealed that these nucleases utilize only a subset of the total potentially

available hydrogen-bonding capacity of the DNA sequence. Thus, HEases tolerate

significant sequence variation both on the side of the protein and the substrate.

This plasticity of the protein-DNA interface greatly facilitates the engineering of

HEases with new specificities, which indeed has been more successful than in the

case of REases. Here, we will only briefly describe the most successful attempts

to engineer the specificity of Type II REases and HEases, with the exception of

engineering of HEases encoded by group II introns, which has been covered in

the preceding section. For more details the reader should refer to excellent recent

reviews (Alves and Vennekohl, 2004; Gimble, 2005).

6.1. Engineering of REases

Based on the analysis of the crystal structure of the R.EcoRV protein-DNA

complex (specificity GAT/ATC, cleavage indicated by “/”), Pingoud and co-workers

randomized selected residues that could evolve new contacts to the DNA, poten-

tially extending the recognition sequence towards XGAT/ATCY (where X and

Y indicate newly recognized, mutually complementary bases). A large-scale in

vitro screen of mutants led to identification of variants with a 25-fold higher

RESTRICTION AND HOMING ENDONUCLEASES 371

preference for the A·T pair flanking wild type recognition sequence versus a

G·C pair(i.e. preferentially cleaving AGAT/ATCT) (Schottler et al., 1998; Wenz

et al., 1998). A similar approach was used to engineer R.BsoBI analogs (speci-

ficity C/RCGYG, where R and Y indicate purine and pyrimidine, respectively)

to recognize only a subset of the wild type recognition sequences. After random-

ization of the single residue (D246) selected by the analysis of the R.BsoBI-DNA

crystal structure, several variants more stringent then the wild type enzyme were

isolated, including the D246A variant showing 70-fold greater binding affinity for

the CCCGGG substrate than for CTCGAG (Zhu et al., 2003).

Another approach developed by Xu and coworkers employs a high-throughput

three-step in vivo selection and screening of variants with potentially new speci-

ficities (Samuelson and Xu, 2002). The mutant variants are initially selected for

survival in a strain expressing a DNA MTase with the desired specificity (which

protects only the “new” target sites). Then, to eliminate variants lacking nuclease

activity selected variants are screened for the ability to induce DNA cleavage

in an indicator strain lacking the DNA MTase. The requirement for a DNA

MTase with a “new” specificity limits the applicability of this system. Nonetheless,

this approach omits the laborious in vitro screening of mutant and allows more

extensive randomization of the target REease gene, which is necessary in the case of

enzymes with unknown structures. It was used successfully to screen for mutants of

R.BstYI (R/GATCY) with specificity increased towards the R.BglII-like sequence

(A/GATCT) (Samuelson and Xu, 2002). Recently R. NotI (GC/GGCCGC) was

engineered in a two-step approach with the first step similar to the in vivo selection

described above, leading to isolation of variants with specificity relaxed towards a

small set of 8 bp substrates (Samuelson et al., 2006). Here, the second step of in

vivo selection aimed at increasing the enzymatic activity of mutant was based on

the cleavage of a conditionally toxic vector carrying the new target sequence.

A conceptually similar screening method called the methylation activity-based

selection (MABS) was used to alter the specificity of Type IIG REase R.Eco57I

(specificity CTGAAG, cleaves at a short distance away from the recognition site).

R.Eco57I comprises the naturally fused, structurally and functionally independent

DNA-binding, MTase, and nuclease domains. The nuclease activity of R.Eco57I

was inactivated by a single substitution in the active site, followed by error-prone

PCR mutagenesis and selection of mutants with the MTase activity specific for new

sequences. In a selected variant the nuclease activity was restored by reversion of

the initial substitution, resulting in an engineered REase that targets a degenerate

sequence CTGRAG (Rimseliene et al., 2003). This variant (Eco57MI) is the first

commercially available REase with engineered sequence specificity.

6.2. Engineering of HEases

Attempts to engineer sequence specificity of HEases have been reported only

recently, but were more successful than in the case of REases. In one of the first

attempts to modify the specificity of I-CreI (the most frequent targets of HEase

372 SKOWRONEK AND BUJNICKI

engineering), the saturating mutagenesis of two amino acid residues critical for

DNA recognition was carried out. The mutants were screened in vivo for their

ability to eliminate a reporter plasmid containing several variants of the altered

I-CreI recognition sequence, leading to isolation of enzymes with specificity shifted

toward the selected sequence; however, the isolated mutants retained the ability

to cleave the wild type target (Seligman et al., 2002). In another HEase, PI-

SceI, the mutagenesis was targeted to the DNA-binding residues within the protein

splicing domain. A bacterial two-hybrid method was used to select PI-SceI variants

from a randomized expression library that exhibited altered DNA recognition

patterns ranging from partial relaxation of specificity to marked shifts in target

site recognition (Gimble et al., 2003). A recently developed approach involves a

high-throughput selection of HEase variants for the ability to induce DSB-induced

homologous recombination in a eukaryotic environment (Chames et al., 2005). It

has been used to analyze a library of I-CreI mutants with three randomized residues

and has led to isolation of hundreds of variants with altered specificities (Arnould

et al., 2006).

Another method involves altering HEase specificity by domain shuffling. For

instance, an active nuclease with modified specificity was created by replacing a

DNA recognition region (a subdomain of the protein splicing domain) of PI-SceI,

with a homologous region of a related protein PI-CtrIP devoid of the nuclease

activity (Steuer et al., 2004). Two groups have independently constructed chimeric

single-chain HEases by fusing the N-terminal nuclease domain of I-DmoI with

a single copy of I-CreI and making different mutations at the domain interface

(Chevalier et al., 2002; Epinat et al., 2003). Expectedly, the resulting chimeric

HEases recognize sequences closely resembling the combination of I-DmoI and

I-CreI half-sites. The domain shuffling approach can be applied not only to

the wild type HEases, but also to the variants engineered by mutagenesis and

screening/selection, potentially leading to a plethora of new specificities.

7. CONCLUSIONS AND FUTURE PROSPECTS

While the steadily increasing number of experimentally characterized REases

and HEases prompted only modest refinements of their functional classifications

compared to those initially proposed, the rapid growth of sequences and structure

databases have lead to a complete revision of the picture of sequence-structure-

function relationships among these enzymes. It is now commonly accepted that

both REases and HEases evolved multiple times, as well as that some families with

vastly different sequences, initially believed to be unrelated, in fact share common

domains and originated from the same ancestral nuclease superfamilies. Thanks

to the combination of crystallographic analyses, sequence comparisons and struc-

tural modeling, and large-scale mutational studies, we now begin to understand the

basis of sequence specificity, both in terms of the evolution at the level of whole

protein families as well as the physical interactions within individual protein-DNA

complexes. Even if structure-guided protein engineering still frequently falls short

RESTRICTION AND HOMING ENDONUCLEASES 373

of providing fully active enzymes with new specificities, progress made recently in

this challenging task (especially for HEases) seems to indicate that this goal may

soon be reached. The availability of a large number of designed nucleases with new

specificities will undoubtedly prompt the development of multiple new applications

of these enzymes and improve our abilities to analyze and manipulate the DNA

both in vitro and in vivo.

ACKNOWLEDGEMENTS

We thank Alfred Pingoud for stimulating discussions and Monika Radlinska for

critical reading of the manuscript. The authors’ research on REases and HEases was

funded by the EMBO/HHMI Young Investigator Award to JMB, by the MEiN (grant

3P04A01124), by the E.U. 6th Framework Programme (contract# MRTN-CT-2005-

019566), and by the NIH (Fogarty International Center grant R03 TW007163-01).

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CHAPTER 22

DNA POLYMERASES FOR PCR APPLICATIONS

RÉGEN DROUIN

∗

, WALID DRIDI AND OUMAR SAMASSEKOU

Service of Genetics, Department of Pediatrics, Faculty of Medicine & Health Sciences, Université

de Sherbrooke, Sherbrooke, Canada

∗

regen.drouin@usherbrooke.ca

1. INTRODUCTION

In 1956 Arthur Kornberg discovered DNA polymerase I of Escherichia coli, the

first enzyme implicated in the replication process to be identified (Kornberg, 1957).

The principal function of DNA polymerases is to copy DNA using one of its strands

as a template and employing small fragments of DNA or RNA as primers for

elongation from the 5' end to the 3'-OH end. In addition to this function, DNA

polymerases are also involved in the maintenance of genome integrity during DNA

replication, DNA repair, homologous recombination, sister chromatid cohesion, cell

cycle checkpoints and the development of the immune system. The dysfunction of

DNA polymerases can cause diseases such as external ophthalmoplegia (Copeland

and Longley, 2003; Ponamarev et al., 2002), Xeroderma pigmentosum (Kannouche

and Stary, 2003) and the process of tumourigenesis (Loeb, 2001).

DNA polymerases have revolutionised molecular biology with their ability to

amplify small amounts of DNA in vitro. Over the last 20 years their use in the

polymerase chain reaction (PCR) has overcome a major limiting factor in daily

medicine i.e. the quantitative problem of the small amounts of DNA available for

testing. These small amounts of DNA can be a single gene, or just part thereof.

Distinct from replication in living organisms, the PCR process can amplify only

relatively short DNA fragments, usually up to 10 kb. However, with the advent of

recombinant polymerases amplified fragments up to 70 kb long can be achieved

(Blanco et al., 1989). Commercial DNA polymerases come from various species,

and they differ in their structures, their catalytic properties such as processivity,

their fidelity of proofreading and their rate of extension of the DNA strand (Bohlke

et al., 2002).

379

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 379–401.

380 DROUIN ET AL.

The PCR technique was developed in 1985 by K. Mullis (Cetus Corporation)

(Mullis et al., 1986), who received the Nobel Prize in Chemistry in 1993 for this

discovery. The original PCR procedure used heating at 94



C in order to denature

DNA - a temperature that destroyed the DNA polymerase used, hence the enzyme

had to be replenished after the heating stage of each cycle. The original PCR

technique was thus very inefficient and labour-intensive since it required much time,

large amounts of DNA polymerase and continual attention throughout the process.

The idea of using the thermoresistant DNA polymerase from Thermus aquaticus,a

thermophilic bacterium described in 1969 (Brock and Freeze, 1969), resolved the

problem and gave birth to the modern PCR technique. Within just a few years of

its invention PCR was being widely used for various purposes, making it one of

the most spectacular developments in the history of molecular biology.

Despite the simplicity of its principle and realization, the PCR technique hides

numerous snares that can compromise the value of the results obtained. Effective

use of PCR requires good laboratory organization, adequate knowledge of each

PCR step, experience and the choice of an appropriate DNA polymerase for the

task intended.

Polymerases commonly used for PCR are obtained from various thermophilic

micro-organisms: Thermus aquaticus (Taq), Pyrococcus furiosus (Pfu polymerase),

Thermococcus litoralis (Wind or Tli polymerase or Vent polymerase) and Thermus

thermophilus (Tth polymerase). The use of a specific polymerase depends on the

type of PCR being undertaken and the nature and the size of template. Polymerase

mixtures have been developed to overcome difficulties associated with the use of

single polymerases in certain types of PCR. Nowadays, the PCR technique is used

on a daily basis in medical and biological research laboratories for numerous tasks

such as the detection of genetic diseases, the identification of genetic fingerprints,

gene cloning, paternity testing and the diagnosis of infectious diseases by detection

of bacteria or viruses (particularly AIDS).

2. STRUCTURE OF DNA POLYMERASES

A number of different DNA polymerases have been discovered recently: five have

been recognized in Escherichia coli, nine in Saccharomyces cerevisiae and sixteen

in humans (Table 1) (Bebenek and Kunkel, 2004). Variations in the amino acid

sequences in the catalytic subunits of the DNA polymerases have been used to

define five families for these enzymes: A, B, C, X and Y (Table 1) (Braithwaite

and Ito, 1993; Burgers et al., 2001). Nevertheless, all DNA polymerases share a

common structural conformation akin to the human right hand comprised of three

distinct domains designated palm, thumb and fingers (Fig. 1) (Franklin et al., 2001;

Lewin, 2004). Conserved sequence motifs in the palm provide the catalytic active

site; correct positioning of the template at the active site is effected by the fingers;

the thumb is involved in binding the DNA and plays a role in processivity of the

enzyme (Steitz, 1998). Conserved regions of these three domains coincide at the

catalytic site.