Structure and Function of an RNA‐Reading Thermostable DNA Polymerase

Abstract

The high substrate specificity of DNA-dependent DNA polymerases is essential for the stability of the genome as well as many biotechnological applications. [1] The discrimination between ribo-and deoxyribonucleotides and between RNA and DNA, particularly in cells, is fundamental since the concentration of ribo moieties by far exceeds that of deoxyribo analogues. Although the selection mechanisms for the incorporation of nucleotides have been investigated intensively for DNA and RNA polymerases, [2] much less is known on how DNA-dependent DNA polymerases discriminate between the different nucleic acid templates (DNA versus RNA). Some viral DNA polymerases (e.g. reverse transcriptases) can utilize both DNA and RNA as a template for nucleic acid synthesis. Crystal-structure analysis of these enzymes, complexed either to an RNA or DNA template, have contributed significantly to our understanding of this process. [3] However, similar structural data of DNA-dependent DNA polymerases that have a poor propensity to process RNA templates are lacking, presumably because the crystallization is hampered by the formation of unstable complexes that leads to structural heterogeneity.Structure analysis of KlenTaq DNA polymerase, a shortened form of Thermus aquaticus DNA polymerase, has added significant contributions to the understanding of how DNA polymerases recognize the cognate substrate, [4] process abasic sites, [5] and non-natural nucleotides. [4d-g] Since our attempts to obtain suitable crystals of KlenTaq complexed to RNA failed, we set out to engineer the enzyme in such a way that it is capable of processing an RNA template more efficiently, which might result in improved crystallization properties. Indeed, we were able to obtain a significantly improved KlenTaq variant from which we obtained structural insights into a DNA-dependent DNA polymerase while processing RNA as a template for the first time. Furthermore, the generated KlenTaq variant turned out to be a thermostable DNA polymerase with significant reverse transcriptase activity, thus resulting in it being a valuable tool for crucial applications in clinical diagnostics and molecular biology, such as transcriptome analysis as well as the detection of pathogens and disease-specific markers.For the generation of an improved enzyme variant the mutation sites of two KlenTaq (KTq) variants M1 (L322M, L459M, S515R, I638F, S739G, E773G) [6] and M747K [7] were recombined by DNA shuffling. Both variants, M1 and M747K, were reported to possess either some reverse transcriptase activity or an expanded substrate spectrum. DNA shuffling was employed since the M1 variant, previously evolved in error-prone PCR, comprises six mutations distributed over the enzyme scaffold, but the individual contributions of the mutations are unknown. We generated a library of 1570 clones to obtain high coverage of all mutation combinations (as described in the Supporting Information). Proteins were expressed in 96-well plates and, after lysis and heat denaturation of the host proteins, use...

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates

“…However, it is less clear how these mutations interact with M‐DNA substrates. Although recent structural studies have increased our understanding of native DNA polymerase recognition of well‐tolerated modified nucleotides, there are still relatively few examples of mutant DNA polymerases recognizing M‐DNA, which is not well‐tolerated by native DNA polymerases . Our engineering efforts suggest that the determinants of modified sugar and modified nucleobase recognition are fairly different and, perhaps orthogonal.…”

XNA Synthesis and Reverse Transcription by Engineered Thermophilic Polymerases

“…Mutations in the same region of the thumb subdomain are critical in HNA, CeNA, LNA, ANA, FANA , and TNA (Larsen et al, 2016) synthesis, and in RT enzymes engineered from DNA polymerases (Blatter et al, 2013;Ellefson et al, 2016). This has been clearly demonstrated in the engineering of an RNA polymerase from a DNA polymerase , where an active-site mutation was required to enable individual NTP incorporation (Tgo: Y409G) and a second mutation in the thumb subdomain was required to enable the binding of the nascent heteroduplex (Tgo: E664K) and synthesis of RNA oligomers longer than 6 nucleotides.…”

Entwicklung einer DNA‐Polymerase für die direkte m6A‐Sequenzierung

“…Das Enzym wurde ausgehend von einer thermostabilen KlenTaq-DNA-Polymerase mit RT-Aktivität( KlenTaqL459M S515R I638F M747K, im Folgenden als RT-KTQ bezeichnet) evolviert. [19] In einem ersten Schritt wurde der Einbau von komplementären und nicht-komplementären Nukleotiden gegenüber m 6 Aund unmodifiziertem Auntersucht. Hierfürw urde ein Primer an jeweils eines von zwei verschiedenen RNAOligonukleotiden (mit derselben Sequenz, aber einem Aoder m 6 Aa nd er Stelle des Einbaus) hybridisiert und dann durch RT-KTQ in Gegenwart eines der vier dNTPs um ein Nukleotid verlängert (Abbildung S1).…”