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Andreas Marx

Publications and source records attributed to Andreas Marx.

28 records · Page 2Linked to original sources
PubMed2002

Implications of active site constraints on varied DNA polymerase selectivity.

DNA polymerase selectivity often varies significantly depending on the DNA polymerase. The origin of this varying error propensity is elusive. It is assumed that DNA polymerases form nucleotide binding pockets that differ in properties such as shape and tightness. We tested this prediction and studied HIV-1 RT by employment of size-augmented nucleotides and site-directed mutagenesis of the enzyme. New valuable insights into the mechanism of DNA polymerase fidelity were obtained. The presented study provides experimental evidence that variations of steric constraints within the nucleotide binding pocket of at least two DNA polymerases cause variations in nucleotide incorporation selectivity. Thus, our results support the concept of active site tightness as a causative in differential fidelity among DNA polymerases.

Binding Sites↗
PubMed2002

Exploring the effects of active site constraints on HIV-1 reverse transcriptase DNA polymerase fidelity.

To examine the concept of polymerase active site tightness as a criteria for DNA polymerase fidelity, we performed pre-steady-state single nucleotide incorporation kinetic analyses with sugar modified thymidine 5'-triphosphate (TTP) analogues and human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The employed TTP analogues (T(R)TP) are modified at the 4'-position of the sugar moiety with alkyl groups, gradually expanding their steric demand. Introduction of a methyl group reduces the maximum rate of nucleotide incorporation by about 200-fold for RT(WT) and about 400-fold for RT(M184V). Interestingly, the affinity of RT for the modified nucleotide is only marginally affected. Increasing the size to an ethyl group leads to further reduction of the rate of incorporation and first effects on binding affinities are observed. Finally, substitution for an isopropyl group results not only in a further reduction of incorporation rates but also in a dramatic loss of binding affinity for the nucleotide analogue. By increasing the steric demand the effects on RT(M184V) in comparison with RT(WT) become progressively more pronounced. Misincorporation of either TTP or T(Me)TP opposite a template G causes additional decline in incorporation rates accompanied by a drastic decrease in binding affinities. This results in relative incorporation efficiencies [(k(pol)/K(d))(incorrect)/(k(pol)/K(d))(TTPcorrect)] of 4.1 x 10(-5) for TTP and 3.4 x 10(-6) for T(Me)TP in case of RT(WT) and 1.4 x 10(-5) for TTP and 2.9 x 10(-8) for T(Me)TP in case of RT(M184V).

Binding Sites↗
PubMed2002

Differential minor groove interactions between DNA polymerase and sugar backbone of primer and template strands.

DNA polymerases are the key enzymes for DNA synthesis involved in DNA replication, recombination, and repair. These enzymes undergo manifold contacts with the primer-template-substrates reaching up to several nucleotide pairs beyond the catalytic centre. To evaluate these enzyme contacts with the DNA substrates we applied novel synthetic steric probes in functional studies. We found that through application of the these probes valuable insights into DNA polymerase function can be gained, which might be useful for the design of new DNA polymerase-based nucleotide variation detection strategies.

DNA↗
PubMed2002

Prolonged intrathecal release of soluble Fas following severe traumatic brain injury in humans.

The mechanisms underlying cell death following traumatic brain injury (TBI) are not fully understood. Apoptosis is believed to be one mechanism contributing to a marked and prolonged neuronal cell loss following TBI. Recent data suggest a role for Fas (APO-1, CD95), a type I transmembrane receptor glycoprotein of the nerve growth factor/tumor necrosis factor superfamily, and its ligand (Fas ligand, FasL) in apoptotic events in the central nervous system. A truncated form of the Fas receptor, soluble Fas (sFas) may indicate activation of the Fas/FasL system and act as a negative feedback mechanism, thereby inhibiting Fas mediated apoptosis. Soluble Fas was measured in cerebrospinal fluid (CSF) and serum of 10 patients with severe TBI (GCS< or =8) for up to 15 days post-trauma. No sFas was detected in CSF samples from patients without neurological pathologies. Conversely, after TBI 118 out of 120 CSF samples showed elevated sFas concentrations ranging from 56 to 4327 mU/ml. Paired serum samples showed above normal (8.5 U/ml) sFas concentrations in 5 of 10 patients. Serum levels of sFas were always higher than CSF levels. However, there was no correlation between concentrations measured in CSF and in serum (r(2)=0.078, p=0.02), suggesting that the concentrations in the two compartments are independently regulated. Also, no correlation was found between sFas in CSF and blood brain barrier (BBB) dysfunction as assessed by the albumin CSF/serum quotient (Q(A)), and concentrations of the cytotoxic cytokine tumor necrosis factor-alpha in CSF, respectively. Furthermore, there was no correlation with two markers of immune activation (soluble interleukin-2 receptor and neopterin) in CSF. Maximal CSF levels of sFas correlated significantly (r(2)=0.8191, p<0.001) with the early peaks of neuron-specific enolase in CSF (a marker for neuronal cell destruction), indicating that activation of the Fas mediated pathway of apoptosis may be in part the direct result of the initial trauma. However, the prolonged elevation of sFas in CSF may be caused by the ongoing inflammatory response to trauma and delayed apoptotic cell death.

Adolescent↗
PubMedDate not supplied

Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants.

This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4' alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4' of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3'-position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.

Alkylation↗