GIS2 has been previously suggested to be a negative regulator of the RAS-cAMP-PKA signalling pathway, which is important for coupling growth and cell cycle progression.
The HU-resistant DNA synthesis detected by flow cytometry could be the result of DNA replication or gene amplification. To distinguish these possibilities, we analysed DNA synthesis by comparative genome hybridization (CGH) on microarrays. Wild-type, elg1Δ, pol30f, and pol30felg1Δ cells were arrested in G1 and synchronously released in the presence of HU for 30 or 60 min. Genomic DNA was extracted, amplified, and hybridized to a whole-genome tiling microarray. Replicated regions were identified as peaks that exhibit increased copy number relative to G1 cells and overlapped confirmed replication origins (from the DNA Replication Origin Database; ; ; indicated as red tracks below the histograms in and ). The distance travelled by 166 replication forks across the genome was computed by measuring the distance from the centre of each replication origin to the left or to the right edge of the peak coordinates of the peak overlapping that origin. The analysis included all forks that had a peak identified in all four strains at both time points, so that the same set of forks was compared across all experiments in analysing the fork distance distributions. We first noted that significant DNA synthesis occurred in wild-type cells in the presence of 0.2 M HU () and that replication fork distance increased between 30 and 60 min (; ), in agreement with the previous observation that DNA replication significantly slows but does not terminate in the presence of HU (). Analysis of the mutant strains revealed two important properties of the HU-resistant DNA synthesis. First, peak height did not exceed a log2 value of 1 indicating that any given locus doubled only once. Second, replicated regions were distributed throughout the genome and largely corresponded to regions containing early-firing replication origins. Together, these data indicate that the HU-resistant DNA synthesis we observed is bona fide DNA replication and not amplification of specific loci. Some replication peaks centered on late-firing origins (as defined by CLB5 dependence; ) were evident, particularly in the pol30felg1Δ cells, consistent with an advanced replication program in these cells (e.g., ARS1506.5; , *).
Deoxyribonucleotide Biosynthesis And Degradation …
Since it was formally possible that the wider replication peaks in the mutants resulted from earlier entry into S phase rather than an increase in the rate of DNA synthesis, we estimated the rates of replication fork progression in the wild-type and mutant cells () by comparing the replication fork distances at 30 min with that at 60 min (). For this analysis, we omitted forks that returned a rate ). The pol30felg1Δ mutant exhibited a significantly higher rate of 261 bp/min (P=3 × 10−9). Thus, the more extensive DNA replication seen in the double mutant reflected an increase in replication fork progression rates. The fork rates for the single mutants were not significantly different from wild type during the time interval analysed. Together, the CGH data indicate that the HU-resistant DNA synthesis in pol30felg1Δ mutants is the result of DNA replication at an increased rate, largely from early-firing replication origins.
08.01.2018 · Nucleotide Bio Synthesis
The integrity of DNA is constantly challenged by environmental and intracellular factors. Failure to safeguard the genome can result in genomic instability, a driving force in carcinogenesis. Conserved signalling cascades mediated by the Mec1 (human ATR) and Rad53 (human Chk1) kinases orchestrate a multifaceted response that enables yeast cells to cope with DNA damage and DNA replication stress. Mec1 and Rad53 facilitate many activities that promote genome integrity in the presence of DNA damage, including the activation of the Dun1 checkpoint kinase, which in turn mediates the upregulation of ribonucleotide reductase (RNR) activity (). RNR catalyses the rate limiting step of deoxyribonucleoside triphosphate (dNTP) synthesis (). Yeast RNR is a tetrameric complex composed of a large and small subunit. Two Rnr1 polypeptides comprise the large subunit, and the small subunit contains Rnr2 and Rnr4 (, ; ). By inhibiting the transcriptional repressor Crt1, Dun1 mediates the transcriptional induction of RNR2 and RNR4 (; ), as well as RNR3, which encodes an alternative component of the large RNR subunit (). Dun1 also triggers the degradation of Sml1 and Dif1, which inhibit RNR activity by directly interfering with catalysis and by nuclear sequestration of the small subunit, respectively (; ; ). Together, these Dun1 activities result in the expansion of intracellular dNTP pools, which promotes DNA repair and cell survival in the presence of genotoxic agents ().
But in case of pyrimidine synthesis pathway, ..
We report the synthesis and characterization of unnatural base pairs bearing propynyl groups, and their use to site-specifically label amplified DNA via Click chemistry. We demonstrate the attachment of a small molecule biotin tag, and for the first time a protein (SH3), to a large, PCR amplified fragment of DNA, as well as the arraying of two proteins on the same duplex.