Organisms utilise cytosine-5 DNA methylation to expand their repertoire of genetic transactions. Structural studies of DNA cytosine-5 methyltransferase have revealed that DNA methyltransferases incorporate nucleotide flipping into their catalytic cycle in order to access the otherwise buried pyrimidine ring from within duplex DNA. Interestingly, substituting the catalytic nucleophile Cys with Gly can produce cytotoxic forms of the bacterial methyltransferases and cause rearrangements in the DNA. In this study the generality of the cytotoxic effect has been studied on both mono and multi-specific methyltransferases. The effect of dimerisation of methyltransferases on the rearrangement event and the specificity of DNA damage have been defined. The involvement of two DNA repair proteins RecA and UmuDC has been studied. The wild type and mutant multispecific methyltransferase (M.SPRI) has been transcribed and translated in vitro and the proteins studied using surface plasmon resonance technique. The experiments described here demonstrate for the first time how a high affinity, catalytically deficient DNA methyltransferase induces error-prone deletions in E.coli.

No description available.

Delicate epigenetic modifications are essential for production of spermatids during spermatogenesis. DNA methyltransferase 3 (DNMT3), the enzymes involved in adding a methyl group to unmodified DNA, contains three members: DNMT3A, DNMT3B and DNMT3L. The latter lacks methyltransferase activity, but was closely associated with spermatogenesis in many reports. According to the presentation of mature spermatids in testis, azoospermia could be separated into obstructive and non-obstructive categories. Non-obstructive azoospermia is spermatogenesis defective, germ cells absent in seminiferous tube is the most serious type. Therefore, we would like to find out if there are differential expression of DNMT3 family transcripts in testes of azoospermia patients from infertility clinic. Using RT-PCR and qPCR, we found only 5 (29.4%) expressed DNMT3L in 17 non-obstructive patients, whereas all 20 obstructive patients expressed. Both groups were similar in expression levels of DNMT3A and DNMT3B. Nuclei of spermatogonia and spermatocyte were the main immunohisto-chemical localization of DNMT3L protein. Lost of germ cells should be the cause of undetectable DNMT3L expression in azoospermia patients. By this founding, it could serve as an indicator for ability of male germ cell culture in further applications of assisted reproduction.

This study determined whether phenotype or genotype tests for thiopurine methyltransferase (TPMT) are cost effective interventions for guiding doses of 6-mercaptopurine in children with acute lymphoblastic leukemia (ALL) compared to standard weight-based dosing. A systematic review of the literature was conducted to assess the accuracy of the TPMT technologies, followed by a cost effectiveness analysis which compared genotype, phenotype and weight-based dosing strategies over a three month time horizon. Both TPMT phenotype and genotype technologies were considered accurate though there is no gold standard. Additionally, included studies were of low methodological quality. Neither of the interventions showed a benefit in survival and both were more costly compared to standard weight-based dosing. At this time there is insufficient evidence to recommend the use of phenotype or genotype testing prior to 6-mercaptopurine therapy to guide initial doses in pediatric ALL patients.

This study determined whether phenotype or genotype tests for thiopurine methyltransferase (TPMT) are cost effective interventions for guiding doses of 6-mercaptopurine in children with acute lymphoblastic leukemia (ALL) compared to standard weight-based dosing. A systematic review of the literature was conducted to assess the accuracy of the TPMT technologies, followed by a cost effectiveness analysis which compared genotype, phenotype and weight-based dosing strategies over a three month time horizon. Both TPMT phenotype and genotype technologies were considered accurate though there is no gold standard. Additionally, included studies were of low methodological quality. Neither of the interventions showed a benefit in survival and both were more costly compared to standard weight-based dosing. At this time there is insufficient evidence to recommend the use of phenotype or genotype testing prior to 6-mercaptopurine therapy to guide initial doses in pediatric ALL patients.

Erlangen, Nürnberg, Universiẗat, Diss., 2007. / Enth. ausserdem 5 Sonderabdr. aus verschiedenen Zeitschr. - Beitr. teilw. dt., teilw. engl.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 58-71).

Universiẗat, Diss., 2004--München.

Techn. University, Diss., 2005--Dresden.

Posttranslational modifications of nucleosomes play a crucial role for the proper functioning of the cell. One of the modifications called methylation is conducted by a family of SET-domain containing proteins called NSD1, NSD2, and NSD3. Recently, more evidence about the involvement of NSD proteins and their mutations in the oncogenesis has emerged. Various studies have found that post-SET domain and basic post-SET extension of NSD proteins are crucial for nucleosome interactions and for conducting enzymatic reactions. In this thesis, I attempt to define the role of post-SET domain basic extension on DNA binding, using nuclear magnetic resonance and isothermal titration calorimetry. Additionally, I have attempted to establish a methodology for obtaining nucleosomes, which could be used in future studies. NMR results showed that the post-SET domain extension is required for DNA to bind to NSD2 and NSD3 methyltransferases. The mutant form of NSD2, E1099K, exhibits stronger binding to DNA than does the wild type of NSD2. NMR results also show that the transplanted version of NSD3 containing the post-SET extension of NSD2 have an affinity similar to that of the NSD2 wild type. The NSD2 transplanted with a post-SET extension of NSD3 has close to 4 times less affinity towards DNA than does NSD2 wild type. The affinity of NSD3 T1232A and wild type could not be obtained, as the proteins could not be expressed in a sufficient amount for ITC experiments. However, the present literature confirms lower affinity of NSD3 (around 4 times less than NSD2) towards nucleosomes. Based on the empirical data and literature-based information, it can be assumed that post-SET domain basic extension determines the binding affinity of a NSD protein towards DNA. Additionally, a successful methodology for obtaining nucleosomes was established for future studies.