PCNA binding proteins in Drosophila melanogaster : the analysis of a conserved PCNA binding domain

The eukaryotic polymerase processivity factor, PCNA, interacts with cell cycle regulatory proteins such as p21^(WAF1/Cip1) and Gadd45, as well as with proteins involved in the mechanics of DNA repair and replication. A conserved PCNA-binding motif is found in a subset of PCNA-interacting proteins, including p21, suggesting that the regulation of these interactions is important for the co-ordination of DNA replication and repair. We have identified several classes of protein which bind to Drosophila PCNA. Two of these proteins contain the consensus PCNA-binding domain: one is the Dacapo protein, a Drosophila homologue of p21^(WAF1/Cip1), and the second is the transposase encoded by the Pogo DNA transposon. A conserved PCNA-binding domain is also present in a human relative of Pogo, named Tigger, suggesting that this domain has a functional role in this class of transposable element. This raises interesting possibilities for a novel method of transposition in which the transposase might be targeted to replicating DNA. Finally, we have investigated the use of this conserved PCNAbinding domain as a predictor of PCNA-binding capacity

PCNA binding proteins in Drosophila melanogaster : the analysis of a conserved PCNA binding domain

The eukaryotic polymerase processivity factor, PCNA, interacts with cell cycle regulatory proteins such as p21^(WAF1/Cip1) and Gadd45, as well as with proteins involved in the mechanics of DNA repair and replication. A conserved PCNA-binding motif is found in a subset of PCNA-interacting proteins, including p21, suggesting that the regulation of these interactions is important for the co-ordination of DNA replication and repair. We have identified several classes of protein which bind to Drosophila PCNA. Two of these proteins contain the consensus PCNA-binding domain: one is the Dacapo protein, a Drosophila homologue of p21^(WAF1/Cip1), and the second is the transposase encoded by the Pogo DNA transposon. A conserved PCNA-binding domain is also present in a human relative of Pogo, named Tigger, suggesting that this domain has a functional role in this class of transposable element. This raises interesting possibilities for a novel method of transposition in which the transposase might be targeted to replicating DNA. Finally, we have investigated the use of this conserved PCNAbinding domain as a predictor of PCNA-binding capacity

One ring to rule them all? Another cellular responsibility for PCNA

To prevent duplication or loss of genomic regions during DNA replication, it is essential that the entire genome is copied precisely once every S phase. Cells achieve this by mutually exclusive regulation of origin firing and licensing. A crucial protein that is involved in origin licensing is chromatin licensing and DNA replication factor 1 (CDT1) and, therefore, activity of this protein must be strictly controlled. Four recent articles have demonstrated that proliferating cell nuclear antigen (PCNA), an essential sliding clamp used in replication and DNA repair, has a crucial role in this process by mediating the proteasomal degradation of CDT1

Experiments and analysis advance R2100 distance sensors used for safety systems of TOMI

In order to increase safety systems reliability of TOMI harvester, it is necessary to use advance R2100 Distance sensors which can scan all kinds of targets and receive data from automatic control system. The Structure and function of R2100 Distance sensors were provided in this paper, In order to determine the best application function of the R2100, effectiveness of R2100 sensors used for TOMI robot with robotic cutting forage were tested and analyzed. For application in precision agricultural engineering automatic control safety systems, static tests were applied with a box, cylinder, cone and person as 4 target samples which were set at different points and lines with each segment at 8° angle within 11 segments, the target samples were set at 0°, 14°, 44°, 74° and 88° angles with the reference of the sensor at centre, respectively, samples represent obstacles such as tractors, telegraph pole, car, and person which were detected and received by TOMI equipped with R2100 Distance sensors. TOMI Robot equipped with R2100 sensors setting at 240, 420 and 850 mm height, respectively, were set location at about 0.2m, 0.3m, 0.5m, 1m, 1.5m, 2m, 2.5m, 3m, 3.5m and then added up to 0.5 m step up to 10 m with the reference of R2100 sensor in semicircle centre, respectively. In dynamistic testing, the target samples were set at the same method and location, and TOMI robot equipped with Advance R2100 sensors was running at speed of 0.8~1.2 m/s from 5 m to the test centre in dynamistic tests. Tests and statistical evaluate results showed that the average R2 on TOMI robot was up to 98.96% in static tests, while the average R2 is up to 98.67% in dynamistic test, and as far as TOMI robot’s safety system, 420 mm height was the best location for scanning all kinds of obstacles. The experiment results showed that the Advance R2100 was accurate sensor for application, it had been carried out on TOMI's intelligence safety systems which more practical and safety working in various fields

Design and evaluate intelligent control safety systems on the TOMI robot

Aimed to design safety systems and evaluate the behaviours about Robot grass cutting named TOMI, the false tree analysis, failure mode effects tree analysis methods were used for review and analysis about the TOMI robot, it would be liability and legislation; TOMI robot management embedded guidelines and knowledge such as Agricultural Engineering, Design and manufacture of agricultural machinery, Mechanic Theory, Mathematics, Electronics, Grass science, Computer science and several software programs; Procedure and Reliability analysis for robots TOMI safety systems are key features,the safety systems of Agricultural Robot such as TOMI should be checked in various working circumstance; With the full consideration of engineering practicability, the solutions to the safety problems of the TOMI robot are promoted, Technology Route and models about TOMI’s safety system were built, Process Management, continual improvement tools and Techniques and effects analysis were built in the new safety systems of TOMI robot. TOMI function measurements such as braking, throttle and pedal force were tested and analysed. TOMI’s Mechanical system, Hydraulics and Electrical Systems were tested for checking safety and evaluated, some sensors and laser such as Distance sensors, SICK, GPS, Dead man handle, safety red button and bumpers were built up and developed the TOMI robot’s new safety systems; To ensure the safety and reliable operation is a system engineering, it is involved to various TOMI robot design, production, operation, adjust, and management; to improve the TOMI robot reliability and reduce the failure frequency was an important way to improve the robot inherent safety; The Evaluation Criteria of Robot grass Cutting DFMEA occurrence may be suggested to use multiple complex technology knowledge and design with more experience. Application built with Microsoft Robot Development studio was run over on the www.webfarming.com. The hazard and risk analysis were detailed about the safety problems of TOMI robot and deeply studied. Development more practical and safety TOMI robot would be carried out at northwest China in the future

PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step

The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5′-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3′-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the KM. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA

PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNA(S228I) mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3(p66) and PolD4(p12). In contrast p21 largely retains the ability to bind PCNA(S228I). This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNA(S228I) binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNA(S228I) for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD

Prevention of Diabetes in NOD Mice by Repeated Exposures to a Contact Allergen Inducing a Sub-Clinical Dermatitis

BACKGROUND: Type 1 diabetes is an autoimmune disease, while allergic contact dermatitis although immune mediated, is considered an exposure driven disease that develops due to epicutaneous contact with reactive low-molecular chemicals. The objective of the present study was to experimentally study the effect of contact allergens on the development of diabetes in NOD mice. As the link between contact allergy and diabetes is yet unexplained we also examined the effect of provocation with allergens on Natural Killer T (NKT) cells, since involvement of NKT cells could suggest an innate connection between the two diseases. METHOD: NOD mice 4 weeks of age were exposed, on the ears, to two allergens, p-phenylenediamine and 2,4-dinitrochlorobenzene respectively, to investigate the diabetes development. The mice were followed for a maximum of 32 weeks, and they were either repeatedly exposed to the allergens or only sensitized a week after arrival. The stimulation of NKT cells by the two allergens were additionally studied in C57BL/6 mice. The mice were sensitized and two weeks later provocated with the allergens. The mice were subsequently euthanized at different time points after the provocation. RESULTS: It was found that repeated application of p-phenylenediamine reduced the incidence of diabetes compared to application with water (47% vs. 93%, P = 0.004). Moreover it was shown that in C57BL/6 mice both allergens resulted in a slight increment in the quantity of NKT cells in the liver. Application of the allergens at the same time resulted in an increased number of NKT cells in the draining auricular lymph node, and the increase appeared to be somewhat allergen specific as the accumulation was stronger for p-phenylenediamine. CONCLUSION: The study showed that repeated topical application on the ears with a contact allergen could prevent the development of diabetes in NOD mice. The contact allergens gave a non-visible, sub-clinical dermatitis on the application site. The preventive effect on diabetes may be due to stimulation of peripheral NKT cells, as shown for provocation with p-phenylenediamine in the C57BL/6 mouse. This epicutaneous procedure may lead to new strategies in prevention of type 1 diabetes in humans

Mechanism of Dinitrochlorobenzene-Induced Dermatitis in Mice: Role of Specific Antibodies in Pathogenesis

Dinitrochlorobenzene-induced contact hypersensitivity is widely considered as a cell-mediated rather than antibody-mediated immune response. At present, very little is known about the role of antigen-specific antibodies and B cells in the development of dinitrochlorobenzene-induced hypersensitivity reactions, and this is the subject of the present investigation.Data obtained from multiple lines of experiments unequivocally showed that the formation of dinitrochlorobenzene-specific Abs played an important role in the development of dinitrochlorobenzene-induced contact hypersensitivity. The appearance of dinitrochlorobenzene-induced skin dermatitis matched in timing the appearance of the circulating dinitrochlorobenzene-specific antibodies. Adoptive transfer of sera containing dinitrochlorobenzene-specific antibodies from dinitrochlorobenzene-treated mice elicited a much stronger hypersensitivity reaction than the adoptive transfer of lymphocytes from the same donors. Moreover, dinitrochlorobenzene-induced contact hypersensitivity was strongly suppressed in B cell-deficient mice with no DNCB-specific antibodies. It was also observed that treatment of animals with dinitrochlorobenzene polarized Th cells into Th2 differentiation by increasing the production of Th2 cytokines while decreasing the production of Th1 cytokines.In striking contrast to the long-held belief that dinitrochlorobenzene-induced contact hypersensitivity is a cell-mediated immune response, the results of our present study demonstrated that the production of dinitrochlorobenzene-specific antibodies by activated B cells played an indispensible role in the pathogenesis of dinitrochlorobenzene-induced CHS. These findings may provide new possibilities in the treatment of human contact hypersensitivity conditions

Subtle Alterations in PCNA-Partner Interactions Severely Impair DNA Replication and Repair

Dynamic switching of PCNA-partner interactions is essential for normal DNA replication and repair in yeast