scholarly journals Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
James R Fuller ◽  
Phoebe A Rice
Keyword(s):  
Reaction Rate ◽  
Dna Bending ◽  
Target Site ◽  
Strand Transfer ◽  
Bacteriophage Mu ◽  
Target Dna ◽  
Order Of Magnitude ◽  
Mu Transpososome ◽  
Transposition Reaction ◽  
Selection Of

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.

scholarly journals Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

Target and Non–target site Mechanisms Confer Resistance to Glyphosate in Canadian Accessions ofConyza canadensis

Weed Science ◽  
2017 ◽  
Vol 66 (2) ◽  
pp. 234-245 ◽  
Author(s):  
Eric R. Page ◽  
Christopher M. Grainger ◽  
Martin Laforest ◽  
Robert E. Nurse ◽  
Istvan Rajcan ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Resistance Mechanisms ◽  
Target Site ◽  
Conyza Canadensis ◽  
Weed Species ◽  
Target Site Resistance ◽  
Selection For ◽  
Simple Sequence ◽  
Growing Region ◽  
Selection Of

Glyphosate-resistant populations ofConyza canadensishave been spreading at a rapid rate in Ontario, Canada, since first being documented in 2010. Determining the genetic relationship among existing Ontario populations is necessary to understand the spread and selection of the resistant biotypes. The objectives of this study were to: (1) characterize the genetic variation ofC. canadensisaccessions from the province of Ontario using simple sequence repeat (SSR) markers and (2) investigate the molecular mechanism (s) conferring resistance in these accessions. Ninety-eightC. canadensisaccessions were genotyped using 8 SSR markers. Germinable accessions were challenged with glyphosate to determine their dose response, and the sequences of 5-enolpyruvylshikimate-3-phosphate synthase genes 1 and 2 were obtained. Results indicate that a majority of glyphosate-resistant accessions from Ontario possessed a proline to serine substitution at position 106, which has previously been reported to confer glyphosate resistance in other crop and weed species. Accessions possessing this substitution demonstrated notably higher levels of resistance than non–target site resistant (NTSR) accessions from within or outside the growing region and were observed to form a subpopulation genetically distinct from geographically proximate glyphosate-susceptible and NTSR accessions. Although it is unclear whether other non–target site resistance mechanisms are contributing to the levels of resistance observed in target-site resistant accessions, these results indicate that, at a minimum, selection for Pro-106-Ser has occurred in addition to selection for non–target site resistance and has significantly enhanced the levels of resistance to glyphosate inC. canadensisaccessions from Ontario.

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

2007 ◽  
Vol 81 (22) ◽  
pp. 12189-12199 ◽  
Author(s):  
Krishan K. Pandey ◽  
Sibes Bera ◽  
Jacob Zahm ◽  
Ajaykumar Vora ◽  
Kara Stillmock ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Dependent Manner ◽  
Strand Transfer ◽  
Viral Dna ◽  
Target Dna ◽  
Immunodeficiency Virus ◽  
Dna Ends ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (∼15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3′-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3′-OH processing. It follows that the IN-viral DNA complex is “trapped” by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.

How effective is KRM-1648 in treatment of disseminated Mycobacterium avium complex infections in beige mice?

1996 ◽  
Vol 40 (2) ◽  
pp. 437-442 ◽  
Author(s):  
B Ji ◽  
N Lounis ◽  
C Truffot-Pernot ◽  
J Grosset
Keyword(s):  
Mycobacterium Avium ◽  
Mycobacterium Avium Complex ◽  
Antimicrobial Effect ◽  
Bacteriostatic Effect ◽  
Dose Selection ◽  
Order Of Magnitude ◽  
The Mean ◽  
Resistant Mutants ◽  
Selection Of

Although the MICs of 3'-hydroxy-5'-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin, or KRM-1648 (KRM), for Mycobacterium avium complex (MAC) were significantly lower than those of other drugs, its in vivo activity was very weak. Beginning 28 days after inoculation, beige mice that had been infected intravenously with 1.87 x 10(7) CFU of MAC 101 were administered KRM alone, clarithromycin (CLARI) alone, or CLARI plus KRM six times weekly for 16 weeks. In contrast to the mice treated with CLARI-containing regimens, the mortality and the mean spleen weights of mice treated with KRM alone (either 10 or 20 mg/kg of body weight per dose) did not differ significantly from those of untreated mice, their numbers of CFU were very much greater than pretreatment values, and multiplication of MAC was only slightly inhibited. Although monotherapy by KRM selected KRM-resistant mutants, the selection was very weak; the mean number of CFU and the frequency of KRM-resistant mutants increased by no more than 1 order of magnitude after 16 weeks of treatment with KRM at 20 mg/kg per dose. Selection of CLARI-resistant mutants was inhibited but not completely prevented by treatment of the mice with CLARI plus KRM. These results indicate that KRM displayed only a weak bacteriostatic effect against the isolate tested in the beige mouse model; its ability to enhance the antimicrobial effect of CLARI or to prevent emergence of CLARI-resistant mutants was very limited.

Locally Available Aggregate and Sediment Production

2003 ◽  
Vol 1819 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Randy B. Foltz ◽  
Mark Truebe
Keyword(s):  
Road Construction ◽  
Forest Road ◽  
Sediment Production ◽  
Magnitude Range ◽  
Long Duration ◽  
Order Of Magnitude ◽  
Equivalent Test ◽  
Basic Characteristics ◽  
Material Tests ◽  
Selection Of

Selection of suitable locally available materials to build strong and durable roads with aggregate surfaces is desired to minimize road construction and maintenance costs and to minimize the detrimental effects of sedimentation. Eighteen aggregates were selected from local sources in Idaho, Oregon, South Dakota, and Washington State. Aggregate was placed in shallow metal frames and compacted to simulate a forest road. The levels of runoff and sediment from a highintensity, long-duration simulated rainstorm were measured. The material tests selected for use in the study included ones that define the basic characteristics of the aggregate, along with a number of tests intended to predict susceptibility to erosion. Each of the tests was statistically evaluated to identify those that best predicted the perceived aggregate quality. The two best indicators of aggregate quality were the results of the sand equivalent test and the P20 portion of the Oregon air degradation test. The best indicator of either runoff or sediment production was the fraction passing the 0.6-mm sieve. Acceptable aggregates, both those of good quality and those of marginal quality, exhibited a 2-order-of-magnitude range in both runoff and sediment production.

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