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41.
Swi1 and Swi3 form the replication fork protection complex and play critical roles in proper activation of the replication checkpoint and stabilization of replication forks in the fission yeast Schizosaccharomyces pombe. However, the mechanisms by which the Swi1-Swi3 complex regulates these processes are not well understood. Here, we report functional analyses of the Swi1-Swi3 complex in fission yeast. Swi1 possesses the DDT domain, a putative DNA binding domain found in a variety of chromatin remodeling factors. Consistently, the DDT domain-containing region of Swi1 interacts with DNA in vitro, and mutations in the DDT domain eliminate the association of Swi1 with chromatin in S. pombe cells. DDT domain mutations also render cells highly sensitive to S-phase stressing agents and induce strong accumulation of Rad22-DNA repair foci, indicating that the DDT domain is involved in the activity of the Swi1-Swi3 complex. Interestingly, DDT domain mutations also abolish Swi1's ability to interact with Swi3 in cells. Furthermore, we show that Swi1 is required for efficient chromatin association of Swi3 and that the Swi1 C-terminal domain directly interacts with Swi3. These results indicate that Swi1 associates with chromatin through its DDT domain and recruits Swi3 to function together as the replication fork protection complex. 相似文献
42.
In animal walking, the gravitational potential and kinetic energy of the center of mass (COM) fluctuates out-of-phase to reduce
the energetic cost of locomotion via an inverted pendulum mechanism, and, in canine quadrupedal walking, up to 70% of the
mechanical energy can be recovered. However, the rate of energy recovery for quadrupedal walking in primates has been reported
to be comparatively lower. The present study analyzed fluctuations in the potential and kinetic energy of the COM during quadrupedal
walking in the Japanese macaque to clarify the mechanisms underlying this inefficient utilization of the inverted pendulum
mechanism in primates. Monkeys walked on a wooden walkway at a self-selected speed, and ground reaction forces were measured,
using a force platform, to calculate patterns of mechanical energy fluctuation and rates of energy recovery. Our results demonstrated
that rates of energy recovery for quadrupedal walking in Japanese macaques were approximately 30–50%, much smaller than those
reported for dogs. Comparisons of the patterns of mechanical energy fluctuation suggested that the potential and kinetic energies
oscillated relatively more in-phase, and amplitudes did not attain near equality during quadrupedal walking in Japanese macaques,
possibly because of greater weight support (reaction force) of the hindlimbs and more protracted forelimbs at touchdown in
the Japanese macaque, two of the three commonly accepted locomotor characteristics distinguishing primates from non-primate
mammals. 相似文献
43.
Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae 总被引:1,自引:0,他引:1
Sugiyama M Yamamoto E Mukai Y Kaneko Y Nishizawa M Harashima S 《Applied microbiology and biotechnology》2006,72(5):947-952
We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MAT
a and MATα strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call ‘chromosome shuffling’, could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae. 相似文献
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Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres 下载免费PDF全文
Bettina A Moser Lakxmi Subramanian Ya‐Ting Chang Chiaki Noguchi Eishi Noguchi Toru M Nakamura 《The EMBO journal》2009,28(7):810-820
To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited. 相似文献
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Hiroshi Fukamachi Hyang Sook Seol Shu Shimada Chikako Funasaka Kanako Baba Ji Hun Kim Young Soo Park Mi Jeung Kim Keiji Kato Mikito Inokuchi Hiroshi Kawachi Jeong Hwan Yook Yoshinobu Eishi Kazuyuki Kojima Woo Ho Kim Se Jin Jang Yasuhito Yuasa 《PloS one》2013,8(8)
Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49fhigh cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49flow cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49fhigh cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49fhigh sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs. 相似文献
49.
Niyant Shah Akira Inoue Seung Woo Lee Kate Beishline Jill M. Lahti Eishi Noguchi 《Experimental cell research》2013
The ChlR1 DNA helicase is mutated in Warsaw breakage syndrome characterized by developmental anomalies, chromosomal breakage, and sister chromatid cohesion defects. However, the mechanism by which ChlR1 preserves genomic integrity is largely unknown. Here, we describe the roles of ChlR1 in DNA replication recovery. We show that ChlR1 depletion renders human cells highly sensitive to cisplatin; an interstrand-crosslinking agent that causes stalled replication forks. ChlR1 depletion also causes accumulation of DNA damage in response to cisplatin, leading to a significant delay in resolution of DNA damage. We also report that ChlR1-depleted cells display defects in the repair of double-strand breaks induced by the I-PpoI endonuclease and bleomycin. Furthermore, we demonstrate that ChlR1-depeleted cells show significant delays in replication recovery after cisplatin treatment. Taken together, our results indicate that ChlR1 plays an important role in efficient DNA repair during DNA replication, which may facilitate efficient establishment of sister chromatid cohesion. 相似文献
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