The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible ...to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Two identical sister copies of eukaryotic chromosomes are synthesized during S phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from ...their synthesis onward by the chromosomal cohesin complex. Replication fork progression is thought to be coupled to establishment of sister chromatid cohesion, facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4, and Ctf18, are found at, and Ctf4 travels along chromosomes with, replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved.
The phosphoinositide (PI)-3-kinase-related kinase (PIKK) family proteins Tel1p and Mec1p have been implicated in the telomere integrity of Saccharomyces cerevisiae. However, the mechanism of ...PIKK-mediated telomere length control remains unclear. Here, we show that Tel1p and Mec1p are recruited to the telomeres at specific times in the cell cycle in a mutually exclusive manner. In particular, Mec1p interacts with the telomeres during late S phase and is associated preferentially with shortened telomeres. We propose a model in which telomere integrity is maintained by the reciprocal association of PIKKs, and Mec1p acts as a sensor for structural abnormalities in the telomeres. Our study suggests a mechanistic similarity between telomere length regulation and DNA double-strand break repair, both of which are achieved by the direct association of PIKKs.
PDE11A is a dual‐substrate, cAMP and cGMP, cyclic nucleotide phosphodiesterase (PDE). Presently four unique variants carrying distinct GAF sequences in the N‐terminal region have been identified. ...While human PDE11A3 and PDE11A4 are known to be specifically expressed in testis and prostate, respectively, PDE11A1 was mainly detected in skeletal muscle. The human PDE11A gene was investigated and revealed to span > 300 kb, contain 23 exons and be mapped on chromosome 2q31. The transcription start sites of PDE11A1, PDE11A3 and PDE11A4 were determined, and the promoter sequences were revealed. Although 5′ flanking genomic regions of PDE11A1 and PDE11A3 had a consensus TATA motif, that of PDE11A4 was a TATA‐less but contained CCAAT box and Sp1‐binding sequence. Interestingly, we found that the exon 2 sequence for N‐terminal region of PDE11A3 encoded an N‐terminal sequence of the cytochrome c pseudogene in an alternate reading frame, and that C‐terminal region of the cytochrome c pseudogene in intron 2 was disrupted by the insertion of Alu repetitive sequence. Furthermore, we examined the exon–intron organization of the PDE2A gene and compared the exon organization among GAF‐PDE family. The exon organization of the PDE11A catalytic domain was very similar to those of PDE5A and PDE6B. However, other GAF‐PDEs, PDE2A and PDE10A, displayed different exon organization from PDE11A although these three PDEs are similar in their amino‐acid sequences to each other. The findings suggested that PDE11A has a common ancestral gene with PDE5A and PDE6s, whereas PDE2A and PDE10A are generated separately from these three GAF‐PDEs.
PDE11A is a dual-substrate, cAMP and cGMP, cyclic nucleotide phosphodiesterase (PDE). Presently four unique variants carrying distinct GAF sequences in the N-terminal region have been identified. ...While human PDE11A3 and PDE11A4 are known to be specifically expressed in testis and prostate, respectively, PDE11A1 was mainly detected in skeletal muscle. The human PDE11A gene was investigated and revealed to span > 300 kb, contain 23 exons and be mapped on chromosome 2q31. The transcription start sites of PDE11A1, PDE11A3 and PDE11A4 were determined, and the promoter sequences were revealed. Although 5' flanking genomic regions of PDE11A1 and PDE11A3 had a consensus TATA motif, that of PDE11A4 was a TATA-less but contained CCAAT box and Sp1-binding sequence. Interestingly, we found that the exon 2 sequence for N-terminal region of PDE11A3 encoded an N-terminal sequence of the cytochrome c pseudogene in an alternate reading frame, and that C-terminal region of the cytochrome c pseudogene in intron 2 was disrupted by the insertion of Alu repetitive sequence. Furthermore, we examined the exon-intron organization of the PDE2A gene and compared the exon organization among GAF-PDE family. The exon organization of the PDE11A catalytic domain was very similar to those of PDE5A and PDE6B. However, other GAF-PDEs, PDE2A and PDE10A, displayed different exon organization from PDE11A although these three PDEs are similar in their amino-acid sequences to each other. The findings suggested that PDE11A has a common ancestral gene with PDE5A and PDE6s, whereas PDE2A and PDE10A are generated separately from these three GAF-PDEs.
A new procedure is proposed to evaluate the 3D crack shape nondestructively with high accuracy by utilizing the direct current electrical potential method. The evaluation procedure is carried out by ...comparing the measured values of electrical potential distribution and those calculated from an analytical method where the crack is modeled as a continuous distribution of 3D double sources. The optimization method is used in order to find accurate solutions. An elliptical embedded crack and a semielliptical surface crack on the back wall in a plate for any aspect ratio of major to minor axes are evaluated especially with respect to location, length and inclination of the crack. The availability of the procedure is verified by some experiments using stainless steels.
A high accuracy nondestructive procedure utilizing the direct current electrical potential method is proposed to evaluate the 3D shape of a crack in a welded plate. An elliptical embedded crack and a ...semi-elliptical surface crack on the back wall are evaluated on location, length and inclination of the crack. The change on electrical potential distribution due to the effect of both electrical and geometrical inhomogeneity of the weld is described, and the availability of the calibration method on the effect is verified for an embedded crack with a reinforcement. The evaluation procedure is carried out by comparing the measured values of the electrical potential distribution, which are corrected by utilizing the calibration method, with the calculated distribution in accordance with optimization method. The availability of the presented procedure is verified by experiments using stainless steels.
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