Every living organism is programmed to reproduce and to pass genetic information to descendants. The information has to be carefully copied and accurately transferred to the next generation.  Therefore organisms have developed the network of conserved mechanisms to survey the protection and precise transfer of the genetic information. Such mechanisms are called checkpoints and they monitor the correct execution of different cell programs. The DNA damage and the replication blocks are surveyed by the conserved Mec1-Rad53 (human ATM/ATR and Chk2, respectively) protein kinase cascade. Mec1 and Rad53 are essential for survival and when activated orchestrate the multiple cellular responses, including the activation of the ribonucleotide reductase (RNR), to the genotoxic stress. RNR is an enzyme producing all four dNTPs - the building blocks of the DNA - and is instrumental for the maintenance both proper concentration and balance of each of dNTPs. The appropriate concentration of the dNTPs should be strictly regulated since inadequate dNTP production can impede many cellular processes and lead to higher mutation rates and genome instability. Hence RNR activity is regulated at many levels, including allosteric and transcriptional regulation and the inhibition at protein level. In our research, we addressed the question of the transcriptional regulation of RNR and the consequences of dNTP malproduction in the terms of the genomic stability. In yeast S. cerevisiae, four genes encode RNR: 2 genes encode a large subunit (RNR1 and RNR3) and 2 genes encode a small subunit (RNR2 and RNR4). All 4 genes are DNA-damage inducible: transcription of RNR2, RNR3 and RNR4 is regulated via Mec1-Rad53-Dun1 pathway by targeting the transcriptional repressor Crt1 (Rfx1) for degradation; on the contrary, RNR1 gene promoter does not contain Crt1-binding sites and is not regulated through the Mec1-Rad53-Dun1 pathway. Instead, we show that intrastrand cross (X)-link recognition protein (Ixr1) is required for the proper transcription of the RNR1 gene and maintenance of the dNTP pools both during unperturbed cell cycle and after the DNA damage. Thus, we identify the novel regulator of the RNR1 transcription. Next, we show that the depletion of dNTP pools negatively affects genome stability in the hypomorphic mec1 mutants: the hyper-recombination phenotype in those mutants correlates with low dNTP levels. By introducing even lower dNTP levels the hyper-recombination increased even further and conversely all the hyper-recombination phenotypes were suppressed by artificial elevation of dNTP levels. In conclusion, we present Ixr1 as a novel regulator of the RNR activity and provide the evidence of role of dNTP concentration in the genome stability.

Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a methyl donor for the re-methylation of homocysteine to methionine. A thermolabile variant of this enzyme, present in approximately 35% of alleles in the North American population, has been associated with cardiovascular disease, neural tube defects, and colon cancer. A cDNA of 2.2kb for human MTHFR has been expressed and results in an active enzyme, but the cDNA and genomic sequences 5' to the ATG start site have not been adequately investigated. The characterization of the 5' region of the human MTHFR gene is reported here. Four additional 5' exonic sequences were localized to a 4kb genomic fragment. The original exon 1 extends directly upstream into a 5' UTR. Three other 5' exons (two with open reading frames) are alternatively spliced into a common splice acceptor site, generating cDNAs with 4 possible 5' ends. The N-terminal peptide sequence of the porcine MTHFR has not been identified in the human sequence suggesting that the missing human coding sequence might be localized further upstream or not conserved across species. A putative chloride ion channel gene (ClC-6) was located in the opposite orientation, at 3.5kb upstream of the original ATG codon, suggesting an overlap with the MTHFR gene and potential co-localization of regulatory elements. A CpG island was identified in the region of a 5' exon (43S) suggesting that a transcription start site and a promoter might be nearby. This work is relevant in understanding the regulation of this important enzyme in folate metabolism.

Methylenetetrahydrofolate reductase (MTHFR) catalyses the conversion of 5, 10-methylenctetrahydrofolate to 5-methyltetrahydrofolate, co-substrate of methionine synthesis. Two types of deficiency have been described for MTHFR. Severe MTHFR deficiency, associated with severe hyperhomocysteinemia and homocystinuria, shows levels of MTHFR activity below 20% of control values. This deficiency has a variable age of onset and shows a wide range of neurological and vascular defects. Mild MTHFR deficiency, with ≈50% enzyme activity and marked enzyme thermolability, has been proposed as a genetic factor in the development of mild hyperhomocysteinemia, a condition associated with neural tube defects and premature vascular disease. / The goal of this thesis was to determine the molecular basis for severe MTHFR deficiency. In order to study this, I isolated a 1.2 Kb partial cDNA encoding human MTHFR I determined that its primary amino acid sequence is homologous to the bacterial enzyme, and encodes the N-terminal catalytic domain of MTHFR. The cDNA was used to isolate a full length 2.27 Kb cDNA, to map the locus to chromosome position 1p36.3, and to isolate genomic clones for human and mouse MTHFR. I characterized the mouse cDNA sequence, as well as the gene structure for both human and mouse genes. I observed 90% identity at the amino acid level, almost identical sizes of exons and location of introns, and similar sizes of introns. The exon sizes ranged from 102bp to 432bp, and intron sizes varied from 250bp to 4.2Kb. / I identified 13 mutations in severe MTHFR deficiency: 10 missense mutations, 1 nonsense mutation, and 2 splicing defects. I determined that a previously-identified common variant (an Ala →Val mutation) was causative of thermolability in severe MTHFR deficiency. I showed a correlation between genotype, residual activity and phenotype in this disease. I also correlated the presence of another genetic defect, Factor V Leiden mutation, with the possible development of thrombo-embolic events in MTHFR deficiency patients. Finally, I analyzed 8 MTHFR mutations in a bacterial expression system. I determined that 4 of these caused significant reduction of activity (below 20% of control). / This thesis contains the first reports of genetic defects in folate metabolism, and a review of available data in severe MTHFR deficiency.

Thesis (PhD)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: C. Martin Lawrence. Includes bibliographical references (leaves 101-112).

Universiẗat, Diss., 2002--Frankfurt (Main).

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The enzyme nitrogenase catalyzes the energy-dependent reduction of dinitrogen to ammonia via biological nitrogen fixation. Nitrogenase is composed of two metalloproteins known as the molybdenum-iron (MoFe) protein and the iron (Fe) protein. The Fe protein, a 60-kDa dimer of the product of the nifH gene, contains a single 4Fe-4S cluster and two Mg-ATP-binding sites, one at each subunit. The Fe protein is the obligate electron donor to the MoFe protein. To date, no other mutual protein has shown to substitute Fe protein in biological fixation, and the NifH is functional only in the presence of the nifessory protein NifM. Interestingly, the protochlorophyllide reductase (ChlL) encoded by the chlL gene of Chlamydomonas reinhardtii shows significant homology and structural similarity with NifH. Previously, our laboratory has shown that the ChlL can substitute the Fe protein in the functioning nitrogenase only in the absence of NifM. We have also shown that the NifM is a PPIase and the Pro-258 located in the C-terminus of NifH is one of the substrates for NifM. Since the least structural homology exists between NifH and ChlL at the C-terminal region, we hypothesized that we can generate a NifM-independent NifH-ChlL chimeric protein by replacing the C-terminus of NifH (that spans the substrate of PPIase) with that of ChlL. To test this idea we created a chimeric construct by replacing the NifH C-terminal region (residues 248-291) with the ChlL C-terminal region (residues 240-294). The chimeric gene was then transformed into the nifM- Azotobacter vinelandii strain AV98. While the wild type nifH could not render a Nif+ phenotype to the nifM- AV98, the chimera could impart Nif+ phenotype to this nifM- strain. This result demonstrated that the NifH-ChlL chimeric protein is NifM-independent.

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy