scholarly journals Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lance D Langston ◽  
Ryan Mayle ◽  
Grant D Schauer ◽  
Olga Yurieva ◽  
Daniel Zhang ◽  
...  
Keyword(s):  
Cell Types ◽  
The Other ◽  
Replication Protein ◽  
Helicase Activity ◽  
Central Channel ◽  
Steric Exclusion ◽  
Dna Strand ◽  
Dna Complex ◽  
Lagging Strand

Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion in which the helicase encircles the tracking strand only and excludes the other strand from the ring. This mode of translocation allows helicases to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG helicase partially encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and greatly stimulates its helicase activity in vitro. Most significantly, Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking DNA strand. We demonstrate that bypass occurs without displacement of the blocks and therefore Mcm10 must isomerize the CMG-DNA complex to achieve the bypass function.

scholarly journals Mcm10 functions to isomerize CMG-DNA for replisome bypass of DNA blocks

2017 ◽  
Author(s):  
Lance D. Langston ◽  
Ryan Mayle ◽  
Grant D. Schauer ◽  
Olga Yurieva ◽  
Daniel Zhang ◽  
...  
Keyword(s):  
Cell Types ◽  
The Other ◽  
Replication Protein ◽  
Duplex Dna ◽  
Replication Forks ◽  
Central Channel ◽  
Steric Exclusion ◽  
Dna Complex ◽  
Lagging Strand

AbstractReplicative helicases of all cell types are rings that unwind DNA by steric exclusion in which the helicase ring only encircles the tracking strand, excluding the other strand outside the ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and enables the replisome to bypass blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex to position only one strand through the central channel. A similar CMG-DNA isomerization is needed at the origin for head-to-head CMGs to bypass one another during formation of bidirectional replication forks.

scholarly journals Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lance Langston ◽  
Mike O’Donnell
Keyword(s):  
Exclusion Process ◽  
The Other ◽  
Dna Unwinding ◽  
Duplex Dna ◽  
Central Channel ◽  
Replicative Helicase ◽  
Steric Exclusion ◽  
Dna Strand

Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand.

404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 317
Author(s):  
T. S. Kim ◽  
Y. Cao ◽  
H. T. Cheong ◽  
B. K. Yang ◽  
C. K. Park
Keyword(s):  
Gene Transfer ◽  
Transfection Efficiency ◽  
The Other ◽  
Control Group ◽  
Dna Uptake ◽  
Alkaline Lysis ◽  
Exogenous Dna ◽  
Other Hand ◽  
Dna Complex

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan's multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P < 0.05) higher in the 105 spermatozoa group than in the other groups (104, 106, and 107 spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 � 0.2, 70.8 � 1.8, and 68.0 � 2.2%, respectively) of storage was significantly (P < 0.05) lower than for fresh spermatozoa (83.3 � 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P < 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 � 2.3%) was significantly (P < 0.05) lower compared to both DNA solution (64.0 � 1.1%) and control (67.8 � 2.3%). The developmental rates of blastocysts were significantly (P < 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 � 1.3 and 11.3 � 0.8%) than in the control group (22.2 � 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 � 12.0%) than in the DNA solution group (38.7 � 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes. This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea

scholarly journals DNA Helicase Activity of PcrA Is Not Required for the Displacement of RecA Protein from DNA or Inhibition of RecA-Mediated Strand Exchange

2007 ◽  
Vol 189 (12) ◽  
pp. 4502-4509 ◽  
Author(s):  
Syam P. Anand ◽  
Haocheng Zheng ◽  
Piero R. Bianco ◽  
Sanford H. Leuba ◽  
Saleem A. Khan
Keyword(s):  
Dna Recombination ◽  
Reca Protein ◽  
Resonance Energy ◽  
Dna Helicase ◽  
Strand Exchange ◽  
Single Pair ◽  
Helicase Activity ◽  
Dna Strand Exchange ◽  
Dna Strand

ABSTRACT PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.

scholarly journals Intermediate frequency magnetic field at 250.8 kHz does not induce DNA damage or “Adaptive Response” in vitro

2019 ◽  
Vol 3 (1) ◽  
pp. 42
Author(s):  
Zsuzsanna Németh ◽  
Erzsébet Laczkovich-Szaladják ◽  
Annamária Brech ◽  
Zsófia Szilágyi ◽  
Györgyi Kubinyi ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Dna Damage ◽  
Ionizing Radiation ◽  
Adaptive Response ◽  
Cell Types ◽  
Intermediate Frequency ◽  
Magnetic Flux Density ◽  
Frequency Magnetic Field ◽  
Dna Strand

The aim of this study was to examine whether intermediate frequency (IF) magnetic field (MF) induces genotoxic effects or play a role in the induction of adaptive response after combined exposure to IF MF and ionizing radiation in leukocytes and in adrenocortical carcinoma cell line H295R. Cells were exposed to 250.8 kHz at the magnetic field strength of 80 A/m (equivalent to 100 µT magnetic flux density) for 20 hours alone, or exposed to IF MF for 20 hours and 24 hours later challenged with ionizing radiation (1.5 Gy X-ray). Evaluation of the DNA damage was performed with alkaline comet assay. Our results showed that there was no significant genotoxic effect of IF MF exposure compared to the controls in both cell types. Furthermore, results did not indicate a statistically significant change in DNA strand breaks in IF MF pre-exposed cells when they were subsequently exposed to 1.5 Gy. Consequently no adaptive response was detected.

scholarly journals UvrD helicase activation by MutL involves rotation of its 2B subdomain

2019 ◽  
Vol 116 (33) ◽  
pp. 16320-16325 ◽  
Author(s):  
Yerdos A. Ordabayev ◽  
Binh Nguyen ◽  
Alexander G. Kozlov ◽  
Haifeng Jia ◽  
Timothy M. Lohman
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Self Assembly ◽  
Kinetic Studies ◽  
Conformational State ◽  
Helicase Activity ◽  
Single Molecule Fret ◽  
Open Conformation ◽  
Dna Complex

Escherichia coli UvrD is a superfamily 1 helicase/translocase that functions in DNA repair, replication, and recombination. Although a UvrD monomer can translocate along single-stranded DNA, self-assembly or interaction with an accessory protein is needed to activate its helicase activity in vitro. Our previous studies have shown that an Escherichia coli MutL dimer can activate the UvrD monomer helicase in vitro, but the mechanism is not known. The UvrD 2B subdomain is regulatory and can exist in extreme rotational conformational states. By using single-molecule FRET approaches, we show that the 2B subdomain of a UvrD monomer bound to DNA exists in equilibrium between open and closed states, but predominantly in an open conformation. However, upon MutL binding to a UvrD monomer–DNA complex, a rotational conformational state is favored that is intermediate between the open and closed states. Parallel kinetic studies of MutL activation of the UvrD helicase and of MutL-dependent changes in the UvrD 2B subdomain show that the transition from an open to an intermediate 2B subdomain state is on the pathway to helicase activation. We further show that MutL is unable to activate the helicase activity of a chimeric UvrD containing the 2B subdomain of the structurally similar Rep helicase. Hence, MutL activation of the monomeric UvrD helicase is regulated specifically by its 2B subdomain.

scholarly journals An in silico and in vitro pipeline for the rapid screening of helicase modulators

2020 ◽  
Vol 25 (1) ◽  
pp. e927
Author(s):  
Eleni Papakonstantinou ◽  
Flora Bacopoulou ◽  
Vasileios Megalooikonomou ◽  
Aspasia Efthimiadou ◽  
Dimitrios Vlachakis
Keyword(s):  
Enzyme Activity ◽  
High Throughput Screening ◽  
Screening Method ◽  
The Other ◽  
Rapid Screening ◽  
Helicase Activity ◽  
Luminescence Signal ◽  
Coated Plate ◽  
Biotin Molecule

To evaluate the potency of potential helicase modulators, we developed an assay of helicase enzyme activity. Using a DNA or RNA biotin labelled oligonucleotide and after the addition of a recombinant helicase, the nucleic acid unwinds, causing the emission of luminescence, which is quantified with a particular antibody. In our assay, one of the DNA oligos was biotinylated, while the other was labelled with digoxygenin (DIG), both in their 5’ termini. The biotin molecule immobilises the DNA duplex on a neutravidin-coated plate and the helicase activity is measured through the unwinding of DNA, due to ATP activation. The subsequent release of DIG-labelled oligos results in a luminescence signal measured with a chemiluminescence antibody. Our goal was to provide a high throughput screening method for potential helicase inhibitors. The method described in this paper has been demonstrated to be fast, easy and reproducible and doesn’t use radiochemicals.

scholarly journals Superiority of Adipose-derived CD34+ Cells over Adipose-derived Stem Cells in Promoting Ischamic Tissue Survival

2021 ◽  
Author(s):  
Yan-Jun Liu ◽  
Tian-Yu Zhang ◽  
Poh-Ching Tan ◽  
Yun Xie ◽  
Pei-Qi Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conditioned Medium ◽  
Umbilical Vein ◽  
Cell Types ◽  
The Other ◽  
Inflammatory Factors ◽  
Tissue Ischemia ◽  
Ischemic Tissue ◽  
Cd34 Cells

Abstract Background: Tissue ischemia usually leads to necrosis and is a threatening condition associated with reconstructive surgery. Promoting the survival of ischemic tissue is critical for improving clinical outcomes. Although various solutions based on stem cells have been reported, there are still limitations to clinical translation. The aim of this study was to develop an effective method to promote the survival of ischemic tissue. Methods: Adipose-derived CD34+ and CD34- cells were obtained by magnetic bead sorting from the stromal vascular faction (SVF). Adipose-derived stem cell (ADSC) were collected by subculture. The angiogenic capacities of CD34+ cells, CD34- cells and ADSC were evaluated in vitro by comparing mRNA and protein expression. Random axial flaps in nude mice were used to evaluate the efficacy of these cells in protecting tissue from necrosis. The effect of these cells in preventing inflammation was also evaluated. Results: Our data suggest that CD34+ cells expressed higher levels of angiogenetic factors and lower levels of inflammatory factors than the other cell types. More vessel branches were formed when human umbilical vein endothelial cells (HUVECs) were treated with conditioned medium from CD34+ cells than conditioned medium from the other cell types. Compared to ADSC, CD34+ cells showed significantly higher efficacy in promoting tissue survival. More CD31+ cells and higher levels of angiogenic factors were observed in tissues from the CD34+ Group than from the other Groups. Lower levels of the proinflammatory factors TNF-α and IL-1b and higher levels of anti-inflammatory factors were found in the CD34+ Group than in the other Groups.Conclusion: Adipose-derived CD34+ cells showed better efficacy in improving ischemic tissue survival than ADSC by reducing tissue inflammation and promoting angiogenesis. CD34+ cells can be obtained easily and may be suitable for clinical applications.

Cell differentiation in isolated inner cell masses of mouse blastocysts in vitro: onset of specific gene expression

Development ◽  
1979 ◽  
Vol 53 (1) ◽  
pp. 367-379
Author(s):  
Marie Dziadek
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Biochemical Marker ◽  
Cell Types ◽  
The Other ◽  
Specific Gene ◽  
Visceral Endoderm ◽  
Secondary Layer ◽  
Parietal Endoderm

Inner cell masses (ICMs) were isolated by immunosurgery from giant blastocysts formedby the aggregation of three morulae. A layer of endoderm cells formed on the outer surface of these primary ICMs in vitro. When this layer was removed by immunosurgery, a secondary endoderm layer formed. Alphafetoprotein (AFP) was used as a biochemical marker tocharacterize visceral endoderm formation in these cultured ICMs. The immunoperoxidase reaction on sections of ICMs cultured for intervals up to 120 h in vitro showed that someprimary endoderm cells contained AFP, but these were always in the minority. The secondary endoderm layer, on the other hand, was composed of predominantly AFP-positive cells.Itis concluded that the primary endoderm contains mainly parietal endoderm cells, while the secondary layer contains visceral endoderm cells. A model is proposed for the consecutive differentiation of parietal and visceral endoderm cell types from the ICM of mouseblastocysts.

scholarly journals Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types

1981 ◽  
Vol 50 (1) ◽  
pp. 299-314
Author(s):  
C.G. Bellows ◽  
A.H. Melcher ◽  
J.E. Aubin
Keyword(s):  
Periodontal Ligament ◽  
Cell Types ◽  
The Other ◽  
Collagen Gels ◽  
Collagen Fibres ◽  
Periodontal Ligament Fibroblasts ◽  
3 Dimensional ◽  
Cell Processes

Monkey periodontal ligament fibroblasts (MPLF cells), human gingival fibroblasts (HGF cells), rat embryonic calvaria cells (REC cells), porcine periodontal ligament epithelial cells (PPLE cells) and rat osteosarcoma 17/2 cells (ROS cells) were incorporated into 3-dimensional collagen gels plated in 60 mm Petri dishes in order: first, to measure the capacity of these cell types to contract; second, to investigate cell-collagen and intercellular relationships during contraction; and third, to define the cellular contribution to tissue contraction in an in vitro system. Measurements at times up to 72 h on 3 ml gels containing 5 × 10(5) cells and with a collagen concentration of 1.20 mg/ml showed that MPLF cells contracted the gels at a significantly greater rate (P less than 0.001) than did the other cell types. In addition, contraction started sooner and was of greater extent than with the other cells. HGF cells contracted the gels more rapidly than REC and PPLE cells, while ROS cells caused no contraction. Several stages of gel compaction could be defined: (1) the attachment of cells to collagen; (2) cellular spreading within the collagen fibre matrix; (3) organization and alignment of collagen fibres by cell processes; (4) cell migration; (5) establishment of intercellular contacts; and (6) the development of a cellular reticular arrangement within the gel and the extension of this arrangement into a 3-dimensional, tissue-like, honeycomb network. Electron microscopic observations on 0.1 ml gels containing MPLF cells showed that, in the early contractile phase, numerous cell processes attached to or enclosed collagen fibrils. These processes contained microfilamentous material and few organelles. In compacted gels, the cells contained an increased amount of distended rough endoplasmic reticulum and Golgi membranes. Since MPLF cells have the capacity for vigorous contraction of the collagen gels and since they develop a reticular, 3-dimensional structure in compacted gels that is reminiscent of the relationship of periodontal ligament fibroblasts to collagen fibres in vivo, it is suggested that they could provide the major force necessary for tooth eruption in vivo. This system also provides a well-defined in vitro model to study the sequential stages that occur during contraction processes.

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