Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

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Using a novel purification scheme and a new assay for detection of DNA kinase activity, a Polymin P-precipitable DNA kinase has been identified and characterized from calf thymus extracts. The DNA kinase activity was able to phosphorylate RNA as well as single-stranded and double-stranded DNA, therefore it has been termed Polymin P-precipitable polynucleotide kinase (PP-PNK). The enzyme had a neutral to alkaline, broad pH optimum that distinguished it from the previously described mammalian DNA kinases that have an acidic pH optimum. The sedimentation coefficient of the enzyme was 3.4-3.8 S, indicating a molecular weight of about 50 kDa. Estimates for the K$ sb{ rm M}$ for ATP were 52 $ mu$M and for the oligonucleotide substrate 8 $ mu$M. The activity was inhibited by pyrophosphate anions and to a lesser extent by sulfate anions. These results differentiate PP-PNK from other mammalian polynucleotide kinases.

Whole cell extracts from fresh calf thymus glands were subjected to Polymin P fractionation and Q Sepharose chromatography. Three peaks of DNA kinase activity, designated SNQI, SNQII and SNQIII, were found in the supernatant fraction. Studies of SNQI have revealed an estimated molecular mass of 50-90 kDa by Superose 12 chromatography, and activity gel analysis following SDS-PAGE identified an active polypeptide of approximately 55 kDa. This enzyme preparation, purified 10,000-fold, phosphorylated 5$ sp prime$-OH-terminated oligodeoxyribonucleotides and double stranded DNA, yet was inactive on an oligoriboadenosine ladder. SNQI functions optimally at an acidic pH in 10 mM MgCl$ sb2$, but is inhibited by both sulfate and pyrophosphate anions. The estimated K$ sb{ rm M}$ values were 2.3 $ mu$M for the oligonucleotide substrate and 11.8 $ mu$M for ATP. Similar to an enzymatic activity previously isolated from rat liver, SNQI is the first bovine preparation to display both 5$ sp prime$ kinase and 3$ sp prime$ phosphatase activities. / Partial purification and characterization of SNQII revealed similarities to SNQI, such as an acidic pH optimum and the presence of 3$ sp prime$ phosphatase activity. DNA kinase activity was also demonstrated in two mammalian cell lines.

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Previous studies suggest a degree of homology between eukaryotic replication, transcription and translation proteins and archaeal ones. Hence, Archaea are considered a simplified model for understanding the complex molecular machinery involved in eukaryotic DNA metabolism. DNA replication in eukaryotic cells is widely studied. In recent years, DNA replication studies expanded on the archaeal DNA replication machinery. P. abyssi was the first archaeon whose genome was fully sequenced. Genome sequencing and comparative genomics have highlighted an MCM-like protein in P. abyssi. In this study, I report the biochemical and structural characterisation of PabMCM. PabMCM is explored as model for understanding more complex eukaryotic MCM proteins and unravelling the biochemical mechanism by which MCM proteins release their helicase activity. The crenarchaeon Sulfolobus solfataricus possesses a simplified toolset for DNA replication compared to Eukaryotes. In particular, S. solfataricus has a subset of the eukaryotic Okazaki fragment maturation factors, among which there are a heterotrimeric DNA sliding clamp, (the proliferating cell nuclear antigen, PCNA), the DNA polymerase B1 (PolB1), the flap endonuclease (Fen1) and the ATP-dependent DNA ligase I (LigI). PCNA functions as a scaffold with each subunit having a specific binding affinity for each of the factors involved in Okazaki fragment maturation. Here, the 3D reconstruction of PCNA in complex with the Okazaki fragment maturation proteins PolB1, LigI and Fen1 is reported.

Indiana University-Purdue University Indianapolis (IUPUI) Indiana University School of Medicine / The islet β cell is central to the maintenance of glucose homeostasis as the β cell is solely responsible for the synthesis of Insulin. Therefore, better understanding of the molecular mechanisms governing β cell function is crucial to designing therapies for diabetes. Pdx1, the master transcription factor of the β cell, is required for the synthesis of proteins that maintain optimal β cell function such as Insulin and glucose transporter type 2. Previous studies showed that Pdx1 interacts with the lysine methyltransferase Set7/9, relaxing chromatin and increasing transcription. Because Set7/9 also methylates non-histone proteins, I hypothesized that Set7/9-mediated methylation of Pdx1 increases its transcriptional activity. I showed that recombinant and cellular Pdx1 protein is methylated at two lysine residues, Lys123 and Lys131. Lys131 is involved in Set7/9 mediated augmented transactivation of Pdx1 target genes. Furthermore, β cell-specific Set7/9 knockout mice displayed glucose intolerance and impaired insulin secretion, accompanied by a reduction in the expression of Pdx1 target genes. Our results indicate a previously unappreciated role for Set7/9 in the maintenance of Pdx1 activity and β cell function. β cell function is regulated on both the transcriptional and translational levels. β cell function is central to the development of type 1 diabetes, a disease wherein the β cell is destroyed by immune cells. Although the immune system is considered the primary instigator of the disease, recent studies suggest that defective β cells may initiate the autoimmune response. I tested the hypothesis that improving β cell function would reduce immune infiltration of the islet in the NOD mouse, a mouse model of spontaneous type 1 diabetes. Prediabetic NOD mice treated with pioglitazone, a drug that improves β cell function, displayed an improvement in β cell function, a reduction in β cell death, accompanied by reductions in β cell autoimmunity, indicating that β cell dysfunction assists in the development of type 1 diabetes. Therefore, understanding the molecular mechanisms involved in β cell function is essential for the development of therapies for diabetes.