Replicate it: Structures of KOD and 9°N DNA polymerases, two enzymes that are widely used to replicate DNA with highly modified nucleotides, were solved at high resolution in complex with primer/template duplex. The data elucidate substrate interaction of the two enzymes and pave the way for further optimisation of the enzymes and substrates.
The capability of DNA polymerases to accept chemically modified nucleotides is of paramount importance for many biotechnological applications. Although these analogues are widely used, the structural basis for the acceptance of the unnatural nucleotide surrogates has been only sparsely explored. Here we present in total six crystal structures of modified 2'-deoxynucleoside-5'-O-triphosphates (dNTPs) carrying modifications at the C5 positions of pyrimidines or C7 positions of 7-deazapurines in complex with a DNA polymerase and a primer/template complex. The modified dNTPs are in positions poised for catalysis leading to incorporation. These structural data provide insight into the mechanism of incorporation and acceptance of modified dNTPs. Our results open the door for rational design of modified nucleotides, which should offer great opportunities for future applications.
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...
The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase  when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase  was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase  and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase  variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.The DNA of every cell in the human body gets damaged more than 50,000 times a day (1). The relation between DNA damage and repair has a significant effect on various cancers, neurological aberrations, and the process of premature aging (2). DNA polymerases are key enzymes that function in maintaining the integrity of the encoded genetic information in DNA replication, DNA repair, DNA recombination, and the bypassing of damages in DNA. Therefore they are central to the aforementioned interplay (3).The most frequent DNA damage observed under physiological conditions are abasic sites resulting from spontaneous hydrolysis of the bond that connects the sugar and the nucleobase in DNA (4). Guanine and adenine nucleobase residues are cleaved most efficiently, resulting in the abasic sugar moiety (AP; see Fig. 1A) with the loss of the genetic information stored in the nucleobase (5). Because these lesions are devoid of genetic information, they are potentially mutagenic. The bulk of this damage is removed by base excision repair pathway, which uses the sister strand to guide incorporation of the right nucleotide in place of the lesion (6, 7). DNA polymerase  acts as the main gap-filling enzyme in base excision repair (8). The enzyme governs for selecting the right nucleotide complementary to the undamaged templating nucleotide (9).DNA polymerases have been grouped in seven different families named A, B, C, D, X, and Y and reverse transcriptases. The grouping depends on sequence homology and structural similarity (10). DNA polymerase  b...
Die hohe Substratspezifität von DNA-abhängigen DNA-Polymerasen ist einerseits für die Stabilität des Genoms, andererseits auch für viele biotechnologische Anwendungen essentiell.[1] Speziell in vivo ist die Diskriminierung zwischen Ribo-und Desoxyribonukleotiden sowie zwischen RNA und DNA wichtig, da die Anzahl der Ribosebausteine die der Desoxyribosebausteine bei weitem übersteigt. Während der Selektionsmechanismus beim Einbau von Nukleotiden durch DNA-und RNA-Polymerasen [2] schon intensiv studiert wurde, ist darüber hinaus wenig bekannt, wie DNA-abhän-gige DNA-Polymerasen zwischen den verschiedenen Nukleinsäuretemplaten (DNA zu RNA) unterscheiden. Einige virale DNA-Polymerasen (z. B. Reverse Transkriptasen) sind in der Lage, DNA und RNA als Templat für die Nukleinsäuresynthese zu verwenden. Die Analyse der Kristallstrukturen dieser Enzyme, im Komplex mit RNA oder DNA als Templat, hat maßgeblich zum Verständnis dieses Prozesses [3] beigetragen. Für DNA-abhängige DNA-Polymerasen, die RNA-Template nur schlecht prozessieren kçnnen, fehlt es allerdings an vergleichbaren strukturellen Daten, was vermutlich durch die Entstehung instabiler Komplexe und einer daraus resultierenden strukturellen Heterogenität, die die Kristallisation erschweren, begründet werden kann.Die Kristallstrukturanalyse der KlenTaq-DNA-Polymerase, einer verkürzten Form der DNA-Polymerase aus Thermus aquaticus, hat erheblich zum Verständnis beigetragen, wie DNA-Polymerasen das natürliche Substrat erkennen [4] sowie abasische Stellen [5] und nicht-natürliche Nukleotide prozessieren.[ [6] In diesem Screening wurden zwei Varianten, RT-KTq 1 und RT-KTq 2, identifiziert und aufgrund ihrer signifikant erhçhten Reverse-Transkriptase-Aktivität gegenüber den parentalen Enzymen und einer minimalen Anzahl an Mutationen weiter charakterisiert. RT-KTq 1 besitzt drei Mutationen (S515R, I638F und M747K), wohingegen RT-KTq 2 zusätzlich zu den Mutationen von RT-KTq 1 eine weitere Mutation aufweist (L459M, Abbildung 1 a). Für Vergleichsstudien wurde über zielgerichtete Mutagenese eine Variante generiert, die alle sieben Mutationen aufweist (KTq M1/M747K). Der KTq-Wildtyp, die parentalen Enzyme M1 und M747K, KTq M1/M747K und RT-KTq 1 und 2 wurden exprimiert, gereinigt, auf die gleiche Proteinkonzentration eingestellt (Abbildung S1) und weiter charakterisiert.
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