Crystallization and preliminary diffraction analysis of the phosphatase domain of PTPN14 in the human papillomavirus E7 binding-defective mutant form

ABSTRACT We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polκ to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polκ in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polκ. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polκ. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polκ in a DNA damage-independent manner. Therefore, association of Polκ with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18 −/− transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18 −/− cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polκ. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polκ to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

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Resistance to β-Lactamase Inhibitor Protein Does Not Parallel Resistance to Clavulanic Acid in TEM β-Lactamase Mutants

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.11.3568-3573.2002 ◽

2002 ◽

Vol 46 (11) ◽

pp. 3568-3573 ◽

Cited By ~ 3

Author(s):

William A. Schroeder ◽

Troy R. Locke ◽

Susan E. Jensen

Keyword(s):

Clavulanic Acid ◽

Double Mutant ◽

Acid Resistance ◽

Site Directed Mutagenesis ◽

Mutant Form ◽

Inhibitor Protein ◽

Wild Type ◽

Defective Mutant ◽

Mutant Forms ◽

Lactamase Inhibitor

ABSTRACT In order to compare patterns of resistance to inhibition by clavulanic acid with patterns of resistance to inhibition by a β-lactamase inhibitor protein (BLIP), R164S, R244S, and R164S/R244S mutant forms of TEM β-lactamase were prepared by site-directed mutagenesis. When kinetic parameters were determined for these mutant and wild-type forms of TEM, the single mutants showed properties that were similar to those in the literature but the double mutant showed properties that were very different. The R164S/R244S double mutant form of TEM retained its resistance to inhibition by clavulanic acid (characteristic of the R244S mutation) but lost all its ability to hydrolyze ceftazidime (characteristic of the R164S mutation). While these characteristics are contrary to those previously reported for an R164S/R244S double mutant, this discrepancy resulted from the use of a defective mutant in the earlier study. Both the single and double mutant forms of TEM remained highly sensitive when tested for inhibition by BLIP, showing only slightly increased resistance compared to that of the wild type; this pattern of resistance is quite different from the pattern of clavulanic acid resistance. The slight increases in resistance to inhibition by BLIP seen in the mutants may have been related to the fact that all of the mutations effected changes in the net charge on the TEM protein that could impede interactions with BLIP.

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Crystallization and preliminary X-ray diffraction analysis of a protease-resistant mutant form of human galectin-8

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309109013554 ◽

2009 ◽

Vol 65 (5) ◽

pp. 512-514 ◽

Cited By ~ 2

Author(s):

Hiromi Yoshida ◽

Nozomu Nishi ◽

Shin-ichi Nakakita ◽

Shigehiro Kamitori

Keyword(s):

Diffraction Analysis ◽

Resistant Mutant ◽

Mutant Form ◽

X Ray Diffraction ◽

X Ray

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Light Optical Diffraction Studies of Macromolecular Ultrastructure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010006828x ◽

1970 ◽

Vol 28 ◽

pp. 258-259

Author(s):

Glen B. Haydon

Keyword(s):

High Resolution ◽

Diffraction Analysis ◽

Diffraction Pattern ◽

Electron Micrographs ◽

Optical Diffraction ◽

Electron Micrograph ◽

Its Analysis ◽

Resolution Electron ◽

Diffraction Patterns

Analysis of light optical diffraction patterns produced by electron micrographs can easily lead to much nonsense. Such diffraction patterns are referred to as optical transforms and are compared with transforms produced by a variety of mathematical manipulations. In the use of light optical diffraction patterns to study periodicities in macromolecular ultrastructures, a number of potential pitfalls have been rediscovered. The limitations apply to the formation of the electron micrograph as well as its analysis.(1) The high resolution electron micrograph is itself a complex diffraction pattern resulting from the specimen, its stain, and its supporting substrate. Cowley and Moodie (Proc. Phys. Soc. B, LXX 497, 1957) demonstrated changing image patterns with changes in focus. Similar defocus images have been subjected to further light optical diffraction analysis.

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Electron Diffraction Analysis of Microtwin Structures in Regrown (III) Silicon

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100054765 ◽

1982 ◽

Vol 40 ◽

pp. 460-461

Author(s):

P. Ling ◽

R. Gronsky ◽

J. Washburn

Keyword(s):

Electron Diffraction ◽

Ion Implantation ◽

Diffraction Analysis ◽

Diffraction Pattern ◽

Direct Consequence ◽

The Other ◽

Electron Diffraction Analysis ◽

Defect Microstructures ◽

Ion Implanted

The defect microstructures of Si arising from ion implantation and subsequent regrowth for a (111) substrate have been found to be dominated by microtwins. Figure 1(a) is a typical diffraction pattern of annealed ion-implanted (111) Si showing two groups of extra diffraction spots; one at positions (m, n integers), the other at adjacent positions between <000> and <220>. The object of the present paper is to show that these extra reflections are a direct consequence of the microtwins in the material.

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A combined ultrastructural and gene molecular research for the relationship between human uterine cervical carcinoma and human papillomavirus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015856x ◽

1990 ◽

Vol 48 (3) ◽

pp. 204-205

Author(s):

Kun Lee ◽

Jingyi Si ◽

Ricai Han ◽

Wei Zhang ◽

Bingbing Tan ◽

...

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Intraepithelial Neoplasia ◽

Hpv Infection ◽

Dot Blot ◽

Homologous Sequences ◽

Normal Cervical Epithelium ◽

The Relationship ◽

Chronic Cervicitis

There are more supports for the view that human papillomavirus (HPV) infection might be an etiological factor in the development of cervical cancer when the association of persistent condylomata is considered. Biopsies from 318 cases with squamous cell carcinoma of uterine cervix, 48 with cervical and vulvar condylomata, 14 with cervical intraepithelial neoplasia (CIN), 34 with chronic cervicitis and 24 normal cervical epithelium were collected from 5 geographic regions of China with different cervical cancer mortalities. All specimens were prepared for Dot blot, Southern blot and in situ DNA-DNA hybridizations by using HPV-11, 16, 18 DNA labelled with 32P and 3H as probes to detect viral homologous sequences in samples. Among them, 32 cases with cervical cancer, 27 with condyloma and 10 normal cervical epitheliums were randomly chosen for comparative EM observation. The results showed that: 1), 192 out of 318 (60.4%) cases of cervical cancer were positive for HPV-16 DNA probe (Table I)

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Computers and diffraction analysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100152197 ◽

1989 ◽

Vol 47 ◽

pp. 44-45

Author(s):

J. A. Eades

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Electron Diffraction ◽

Diffraction Analysis ◽

Electron Diffraction Analysis ◽

Image Simulation ◽

Correct Interpretation ◽

High Profile ◽

High Resolution Images ◽

Image Simulations

For well over two decades computers have played an important role in electron microscopy; they now pervade the whole field - as indeed they do in so many other aspects of our lives. The initial use of computers was mainly for large (as it seemed then) off-line calculations for image simulations; for example, of dislocation images.Image simulation has continued to be one of the most notable uses of computers particularly since it is essential to the correct interpretation of high resolution images. In microanalysis, too, the computer has had a rather high profile. In this case because it has been a necessary part of the equipment delivered by manufacturers. By contrast the use of computers for electron diffraction analysis has been slow to prominence. This is not to say that there has been no activity, quite the contrary; however it has not had such a great impact on the field.

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