scholarly journals Supporting evidence for DNA shearing as a mechanism for origin unwinding in eukaryotes

2019 ◽  
Author(s):  
Lance D. Langston ◽  
Roxana Georgescu ◽  
Mike E. O’Donnell
Keyword(s):  
Single Strand ◽  
Supporting Evidence ◽  
Duplex Dna ◽  
Shearing Force ◽  
Dna Shearing

ABSTRACTOur earlier study demonstrated that head-to-head CMGs that encircle duplex DNA and track inward at origins, melt double-strand (ds) DNA while encircling the duplex by pulling on opposite strands and shearing DNA apart (Langston and O’Donnell (2019) eLife 9, e46515). We show here that increasing the methylphosphonate neutral DNA from 10 nucleotides in the previous report, to 20 nucleotides, reveals that CMG encircling duplex DNA only interacts with the tracking strand compared to the non-tracking strand. This significantly enhances support that CMG tracks on duplex DNA by binding only one strand. Furthermore, EMSA assays using AMPPNP to load CMG onto DNA shows a stoichiometry of only 2 CMGs on an origin mimic DNA, containing a 150 bp duplex with two 3 prime single-strand (ss) tails, one on each end, enabling assay of dsDNA unwinding by a shearing force produced by only two head-to-head CMGs. The use of non-hydrolysable AMPPNP enabled a preincubation for CMG binding the two 3 prime tailed origin mimic DNA, and gave robust unwinding of dsDNA by head-to-head CMG-Mcm10’s. With this precedent, it is possible to envision that the cell may utilize opposing dsDNA motors to unwind DNA for other types of DNA transactions besides origin unwinding.

The yeast alpha 1 and MCM1 proteins bind a single strand of their duplex DNA recognition site

1992 ◽  
Vol 12 (8) ◽  
pp. 3573-3582
Author(s):  
E J Grayhack
Keyword(s):  
Yeast Cell ◽  
Binding Sites ◽  
Dna Recognition ◽  
Binding Activity ◽  
Single Strand ◽  
Duplex Dna ◽  
Cell Type ◽  
Recognition Sequence ◽  
Binding Properties ◽  
Constitutive Factor

The yeast cell type regulator alpha 1 cooperates with a constitutive factor, MCM1 protein, to recognize the promoter and activate transcription of several alpha-specific genes. I show here that the alpha 1 and MCM1 proteins bind specifically to one of the two strands of their recognition sequence. This single-strand-binding activity shares several characteristics with the duplex-binding properties of these proteins: (i) the MCM1 protein binds alone to single-stranded and duplex sequences of both the alpha-specific (P'Q) and a-specific (P) binding sites; (ii) the alpha 1 protein requires both the MCM1 protein and the Q sequence to bind either single-stranded or duplex DNA; (iii) the alpha 1 protein stimulates binding of the MCM1 protein to both single-stranded and duplex DNAs; and (iv) the affinities of the proteins for single-stranded and duplex DNAs are comparable.

scholarly journals The yeast alpha 1 and MCM1 proteins bind a single strand of their duplex DNA recognition site.

1992 ◽  
Vol 12 (8) ◽  
pp. 3573-3582 ◽  
Author(s):  
E J Grayhack
Keyword(s):  
Yeast Cell ◽  
Binding Sites ◽  
Dna Recognition ◽  
Binding Activity ◽  
Single Strand ◽  
Duplex Dna ◽  
Cell Type ◽  
Recognition Sequence ◽  
Binding Properties ◽  
Constitutive Factor

The yeast cell type regulator alpha 1 cooperates with a constitutive factor, MCM1 protein, to recognize the promoter and activate transcription of several alpha-specific genes. I show here that the alpha 1 and MCM1 proteins bind specifically to one of the two strands of their recognition sequence. This single-strand-binding activity shares several characteristics with the duplex-binding properties of these proteins: (i) the MCM1 protein binds alone to single-stranded and duplex sequences of both the alpha-specific (P'Q) and a-specific (P) binding sites; (ii) the alpha 1 protein requires both the MCM1 protein and the Q sequence to bind either single-stranded or duplex DNA; (iii) the alpha 1 protein stimulates binding of the MCM1 protein to both single-stranded and duplex DNAs; and (iv) the affinities of the proteins for single-stranded and duplex DNAs are comparable.

scholarly journals Conformational Control of Thymine Photodimerization in Single-Strand and Duplex DNA Containing Locked Nucleic Acid TT Steps

2010 ◽  
Vol 132 (44) ◽  
pp. 15831-15831 ◽  
Author(s):  
Mahesh Hariharan ◽  
Martin McCullagh ◽  
George C. Schatz ◽  
Frederick D. Lewis
Keyword(s):  
Nucleic Acid ◽  
Single Strand ◽  
Locked Nucleic Acid ◽  
Duplex Dna ◽  
Conformational Control

Single-strand breaks in duplex DNA of coliphage T7 as demonstrated by electron microscopy

1965 ◽  
Vol 14 (1) ◽  
pp. 271-IN13 ◽  
Author(s):  
David Freifelder ◽  
Albrecht K. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Single Strand ◽  
Duplex Dna ◽  
Strand Breaks ◽  
Single Strand Breaks

scholarly journals Interaction of RecBCD Enzyme with DNA at Double-Strand Breaks Produced in UV-Irradiated Escherichia coli: Requirement for DNA End Processing

1998 ◽  
Vol 180 (21) ◽  
pp. 5639-5645 ◽  
Author(s):  
Brigitte Thoms ◽  
Wilfried Wackernagel
Keyword(s):  
Excision Repair ◽  
Uv Light ◽  
Single Strand ◽  
Duplex Dna ◽  
Dna Fragments ◽  
Chromosomal Dna ◽  
Phage T4 ◽  
Recbcd Enzyme ◽  
Dna Ends ◽  
Activity Decrease

ABSTRACT The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m2). The activity decrease was seen by an increase in survival of phage T42 − of about 200-fold (phage T42 − has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb +) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, andsbcCD, particularly in the recJ sbcCD andxonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.

scholarly journals Kinetic Characterization of Single Strand Break Ligation in Duplex DNA by T4 DNA Ligase

2011 ◽  
Vol 286 (51) ◽  
pp. 44187-44196 ◽  
Author(s):  
Gregory J. S. Lohman ◽  
Lixin Chen ◽  
Thomas C. Evans
Keyword(s):  
Strand Break ◽  
Single Strand ◽  
Dna Ligase ◽  
Duplex Dna ◽  
Kinetic Characterization ◽  
Single Strand Break ◽  
T4 Dna Ligase

Single Strand Targeted Triplex Formation: Strand Displacement of Duplex DNA by Foldback Triplex-Forming Oligonucleotides

1995 ◽  
Vol 13 (3) ◽  
pp. 483-491 ◽  
Author(s):  
Ekambar R. Kandimalla ◽  
Adrienne N. Manning ◽  
Sudhir Agrawal
Keyword(s):  
Single Strand ◽  
Duplex Dna ◽  
Strand Displacement ◽  
Triplex Formation

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions

Gene ◽  
1979 ◽  
Vol 7 (3-4) ◽  
pp. 303-316 ◽  
Author(s):  
R.Stephen Lloyd ◽  
Charles W. Haidle ◽  
Donald L. Robberson
Keyword(s):  
Single Strand ◽  
Duplex Dna
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