Abstract
DNA polymerases need to be engineered to achieve optimal performance for biotechnological applications, which often require high fidelity replication when using modified nucleotides and when replicating difficult DNA sequences. These tasks are achieved for the bacteriophage T4 DNA polymerase by replacing leucine with methionine in the highly conserved Motif A sequence (L412M). The costs are minimal. Although base substitution errors increase moderately, accuracy is maintained for templates with mono- and dinucleotide repeats while replication efficiency is enhanced. The L412M substitution increases intrinsic processivity and addition of phage T4 clamp and single-stranded DNA binding proteins further enhance the ability of the phage T4 L412M-DNA polymerase to replicate all types of difficult DNA sequences. Increased pyrophosphorolysis is a drawback of increased processivity, but pyrophosphorolysis is curbed by adding an inorganic pyrophosphatase or divalent metal cations, Mn2+ or Ca2+. In the absence of pyrophosphorolysis inhibitors, the T4 L412M-DNA polymerase catalyzed sequence-dependent pyrophosphorolysis under DNA sequencing conditions. The sequence specificity of the pyrophosphorolysis reaction provides insights into how the T4 DNA polymerase switches between nucleotide incorporation, pyrophosphorolysis and proofreading pathways. The L-to-M substitution was also tested in the yeast DNA polymerases delta and alpha. Because the mutant DNA polymerases displayed similar characteristics, we propose that amino acid substitutions in Motif A have the potential to increase processivity and to enhance performance in biotechnological applications. An underlying theme in this chapter is the use of genetic methods to identify mutant DNA polymerases with potential for use in current and future biotechnological applications.
Highlights
The remarkable advances made in molecular biology in the last 40 years are dependent to a large extent on DNA polymerasedependent methods
The phage T4 exonuclease-deficient L412M-DNA polymerase is proven to be an excellent DNA sequencing enzyme especially when combined with processivity factors and singlestranded DNA (ssDNA) binding protein; this is the basis of FIDELITYTM
While the T4 L412M-DNA polymerase remains an attractive candidate as a DNA sequencing enzyme, other DNA polymerases may have additional desirable properties that would profit by modifications to the Motif A sequence
Summary
The remarkable advances made in molecular biology in the last 40 years are dependent to a large extent on DNA polymerasedependent methods. Researchers who pioneered the use of DNA polymerases for DNA sequencing, PCR and site-directed mutagenesis were awarded Nobel prizes for their ground-breaking achievements, but DNA polymerases play key roles in numerous additional indispensable methods including DNA labeling, cloning, whole genome amplification, and diagnostic techniques. Researchers, recognized from the earliest days that some DNA polymerase activities are counter-productive for in vitro applications. For example, can degrade primers and the newly synthesized DNA, yet some exonucleolytic proofreading activity is needed for high fidelity DNA replication. Other DNA polymerase activities need to be modified, for example the ability to utilize non-standard nucleotides and to replicate “difficult” DNA templates with simple repeats or sequences that are excessively rich in A+T or G+C base pairs. DNA polymerases from several organisms have been subject to extensive engineering in order to remove or curb unwanted activities and to modify or enhance others that are required for optimal performance in vitro (see several chapters in this volume and reviews by Hamilton et al, 2001; Reha-Krantz, 2008)
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