Rudimentary G-Quadruplex-Based Telomere Capping In Saccharomyces Cerevisiae

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3\u27 overhang inhibits 5\u27-\u3e 3\u27 resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo

Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length

Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR–protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein

A non-canonical DNA structure is a binding motif for the transcription factor SP1 in vitro

SP1 is a ubiquitous transcription factor that is involved in the regulation of various house-keeping genes. It is known that it acts by binding to a double-stranded consensus motif. Here, we have discovered that SP1 binds also to a non-canonical DNA structure, a G-quadruplex, with high affinity. In particular, we have studied the SP1 binding site within the promoter region of the c-KIT oncogene and found that this site can fold into an anti-parallel two-tetrad G-quadruplex. SP1 pull-down experiments from cellular extracts, together with biophysical binding assays revealed that SP1 has a comparable binding affinity for this G-quadruplex structure and the canonical SP1 duplex sequence. Using SP1 ChIP-on-chip data sets, we have also found that 87% of SP1 binding sites overlap with G-quadruplex forming sequences. Furthermore, while many of these immuoprecipitated sequences (36%) even lack the minimal SP1 consensus motif, 5′-GGGCGG-3′, we have shown that 77% of them are putative G-quadruplexes. Collectively, these data suggest that SP1 is able to bind both, canonical SP1 duplex DNA as well as G-quadruplex structures in vitro and we hypothesize that both types of interactions may occur in cells

DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution

In this PhD thesis, several projects about the functional analysis and recruitment of mutated DNA polymerases for improved biotechnological applications were investigated. Discrimination of incorrect pairing single nucleotides is of fundamental importance for the enzyme-aided detection of single nucleotide variations (single nucleotide polymorphisms (SNPs)). It could be demonstrated that both chemically modified primer probes which are thiolated at the 2-position of thymidine as well as mutated DNA polymerases were able to increase single-nucleotide discrimination.Based on these findings a DNA chip based system for the multiplex detection of single nucleotide polymorphisms (SNPs) was established. For that purpose, a mutated DNA polymerase from Pyrococcus furiosus with improved single nucleotide discrimination properties is used for selective microarrayed primer extensions. It is shown that the mutated DNA polymerase in combination with unmodified primer strands fulfils the demands on solid support and obviates the need for chemical modifications of the primer probes as required before. The system depicted herein could provide the basis for further advancements in microarrayed nucleic acid diagnostics using tailor-made enzymes.Until now, all reported methods for DNA polymerase evolution are restricted to a single enzyme property, for example, increased selectivity or the ability to efficiently process DNA lesions. Thus, a new microarrayed device was developed to overcome these obvious limitations that allow the multiplexed screening of several enzyme features in parallel:The approach is based on the spatial separation of different covalently attached DNA substrates on a glass slide and their selective addressing by oligonucleotide hybridization. This system, termed oligonucleotide-addressing enzyme assay (OAEA), enables multiplexed simultaneous profiling of DNA polymerases in nanoliter volumes in terms of their different properties. OAEA can be used for the simultaneous and multiplexed profiling of several enzyme features with high throughput. Additionally, other DNA-modifying enzymes like ligases and endonucleases can be included in multiplex directed evolution approaches using OAEA. As a first successful demonstration it was used to identify enzymes with altered properties out of a library of DNA polymerase mutants.A functional chimeric DNA polymerase could be obtained by fusion of a wild-type 5 ́- 3 ́nuclease domain with a recently described N-terminally shortened DNA polymerase from Thermus Aquaticus, which exhibits a significantly increased reverse transcription activity. The new enzyme (named as Taq M1) was created to improve RNA pathogen detection systems for pathogens like Dobrava viruses. It could be demonstrated that the fusion of polymerase- and 3 ́nuclease-domain to constitute Taq M1 has no effect on the originally polymerase- and nuclease function and activities. Additionally, Taq M1 was used in applied TaqMan RNA detection assays: Without optimisation of reaction conditions Taq M1 provided detection sensitivities compared to commercially available one-step RT PCR systems, which are based on enzyme blends. Taq M1 is highly recommended for the use of one-step RT PCR, especially if high transcription temperatures are desired to melt stable secondary structures of RNA targets.In my last project, functional studies were conducted with the N-terminally shortened DNA polymerase from Thermus Aquaticus (KlenTaq). Recently obtained crystal structures of KlenTaq in complex with both an abasic site harbouring template and a blunt-ended primer template substrate (Schnur et al. and personal communication with S.Obeid), revealed that amino acid tyrosine 671 plays an important role in the template-less selection of the incorporated nucleotide. Tyrosine 671 thereby mimics the steric constraints of a pyrimidine template base resulting in the favoured incorporation of purine bases (A and G). Mutation of tyrosine into alanine (Y671A) results in a dramatic drop of catalytic activity. Mutation of the aromatic tyrosine into the also aromatic but steric more demanding tryptophane results in the favoured incorporation of pyrimidine bases (T and C). These findings could be proved by single nucleotide incorporation studies and enzyme kinetic measurements

Engineered DNA Polymerases in Biotechnology

DNA polymerases are the enzymes that catalyse all DNA synthesis in Nature often with astounding speed and accuracy. Consequently, their features as molecular machines are exploited in a wide range of biotechnological applications. Some features are highlighted in the following. For example, DNA polymerases are useful enzymes to detect genomic alterations that can lead to the development of certain diseases such as cancer or to promote toxic side effects of drugs. Methods for the detection of single-nucleotide polymorphisms, copy-number variations and somatic copy-number alterations are important for the realisation of personalised medicine.[1 3] Additionally, new DNA sequencing technologies aim to achieve the $ 1000 genome that might further drive a new era of specific pharmaceutical treatments and diseaseprevention strategies based on an individual s genome.[4 7] In many DNA diagnostic and sequencing methods, the accurate action of a DNA polymerase to incorporate the right nucleotide with high selectivity, according to the Watson Crick rule, is crucial

New Strategies for DNA Polymerase Library Screening

Engineered enzymes are of increasing importance for a plethora of biotechnical applications. Especially DNA polymerases are workhorses in biochemical technologies in particular the polymerase chain reaction (PCR), cDNA cloning procedures, genome sequencing and in diagnostic applications. DNA polymerase mutant libraries can be used for the screening of non-standard reaction conditions or substrates e.g. the efficient amplification of difficult templates like ancient DNA. We are convinced that these fascinating enzymes can be optimized and costum-made for a specific application to result in more robust and reliable systems. To our knowledge, all known screening methods for DNA polymerase mutants are focused and thus limited to the screening of a single reaction or one new function. We developed improved strategies for multiplexed DNA polymerase screening that will be presented

Fingerabdrücke von DNA-Polymerasen : mehrfache simultane Enzym-Charakterisierung auf DNA-Arrays

DNA-Polymerasen kommen bei einer ganzen Fülle von biotechnologischen Anwendungen zum Einsatz, insbesondere bei der Polymerasekettenreaktion (PCR), genetischen Klonierungen, Genomsequenzierungen und bei diagnostischen Anwendungen.[1] Für Klonierungen sind hoch prozessive und möglichst fehlerfreie DNA-Polymerasen erwünscht, da diese zu kürzeren Verlängerungszeiten und zu einer robusteren Amplifikation mit großer Ausbeute führen. Eine höhere Genauigkeit von DNA-Polymerasen könnte Genomsequenzierungen und diagnostische Anwendungen verlässlicher machen.[2] Die Amplifikation von prähistorischen DNAProben erfordert DNA-Polymerasen mit einem erweiterten Substratspektrum, damit typische DNA-Schäden effizient überlesen werden können.[3] Um die Effizienz von forensischen DNA-Tests zu erhöhen, braucht man DNA-Polymerasen, die resistent gegen Inhibitoren aus Blut- und Erdproben sind und somit eine PCR ohne vorherige DNA-Reinigung möglich machen.[4] Weitere Verbesserungen von DNA-Polymerasen sind z.B. notwendig, um den Anforderungen von DNA-Sequenzierungen einzelner Moleküle zu genügen, die auf dem effizienten Einbau modifizierter Nucleotide beruhen.[5] Insgesamt werden dringend maßgeschneiderte, künstliche DNA-Polymerasen benötigt, die zu robusteren und spezifischeren Reaktionssystemen führen

Taking Fingerprints of DNA Polymerases : Multiplex Enzyme Profiling on DNA Arrays

DNA polymerases are used in a plethora of biotechnical applications, especially in the polymerase chain reaction (PCR), genetic cloning procedures, genome sequencing, and diagnostic methods.[1] Highly processive and accurate DNA polymerases are desired for cloning procedures in order to give shorter extension times as well as more robust and highyield amplification. A higher DNA polymerase fidelity may increase the reliability of genome sequencing and diagnostic systems.[2] Amplification of ancient DNA samples requires DNA polymerases with an increased substrate spectrum to efficiently overcome typical DNA lesions.[3] To enhance the efficiency of forensic DNA testing, DNA polymerases resistant to inhibitors from blood and soil allow PCR without prior DNA purification.[4] Further improvements of DNA polymerases are required, for example, to meet the requirements of real-time DNA single-molecule sequencing, which relies on the ability ofDNApolymerases to efficiently process modified nucleotides.[5] Overall, customized and artificially engineered DNA polymerases that lead to more robust and specific reaction systems are urgently needed

Variants of a Thermus aquaticus DNA polymerase with increased selectivity for applications in allele- and methylation-specific amplification

The selectivity of DNA polymerases is crucial for many applications. For example, high discrimination between the extension of matched versus mismatched primer termini is desired for the detection of a single nucleotide variation at a particular locus within the genome. Here we describe the generation of thermostable mutants of the large fragment of Thermus aquaticus DNA polymerase (KlenTaq) with increased mismatch extension selectivity. In contrast to previously reported much less active KlenTaq mutants with mismatch discrimination abilities, many of the herein discovered mutants show conserved wild-type-like high activities. We demonstrate for one mutant containing the single amino acid exchange R660V the suitability for application in allele-specific amplifications directly from whole blood without prior sample purification. Also the suitability of the mutant for methylation specific amplification in the diagnostics of 5-methyl cytosines is demonstrated. Furthermore, the identified mutant supersedes other commercially available enzymes in human leukocyte antigen (HLA) analysis by sequence-specific primed polymerase chain reactions (PCRs)

Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription : a ‘zero-step’ RT-qPCR protocol

We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a downregulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression. We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our ‘zero-step’ RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5 h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.publishe