Figure 9. Effect of the wild type and mutant forms of RTP on replication fork progression in vitro. A, diagram showing the replication assay. The template that has a Col E1 ori and the Ter 1 site of B. subtilis in either orientation was replicated in the presence of RTP and resolved in...
Our work indicates that conformational penalties are a major determinant of protein–DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11....
(Figure 22.3). It is an importantlaboratory procedurefor producing labeled DNA that can be used as probes; simply by carrying out the reaction in the presence of radioactive- or fluorescence-labeled nucleotides, an unlabeled DNA molecule with nicks in both strands can be converted to a labeled...
The primer−template was annealed and added to 20 μL of reaction mixture containing 50 mM Tris-HCl (pH 8.6), 50 mM KCl, 2 mM MgCl2, 20 μM each of three unlabeled dNTPs, 2 μCi of [α-32P] dATP and 3 μg of the mitochondria. After incubation at 37 °C ...
DNA polymerase (pol) processivity, i.e., the bases a polymerase extends before falling off the DNA, and activity are important for copying difficult DNA sequences, including simple repeats. Y-family pols would be appealing for copying difficult DNA and i
We recently used smFRET to characterize the mechanism of double flap (DF) substrate recognition by DNA replication and repair Flap Endonuclease 1 (FEN1)25. Briefly, FEN1 cleaves excess 5′-flaps from the DNA by threading the 5′-flap into a capped helical gateway25,26. In-vitro, DF is ...
Possible contributing factors include disease-causing mutations in other nuclear genes; altered interactions with other mtDNA replication proteins, such as the mitochondrial Twinkle helicase or the single-stranded DNA-binding protein; the inherited level of somatic mtDNA heteroplasmy; epigenetic factors; and...
A, diagram illustrating the samples analyzed by iPOND-MS. Cells labeled with EdU were processed without the click reaction reagent (Negative control) or treated with thymidine (Thd) for 1 h (Thd chase negative control) prior to iPOND. rxn, reaction. The active replication fork sample was colle...
The DNA screening sequence is preferably from the HSV origin of replication, and the binding protein is preferably UL9. The invention also includes, a method for altering the binding characteristics of a DNA-binding protein to a duplex DNA. In the method, a binding site for the DNA-binding ...
(with 4 μM DdrA protein), except that they have been challenged with a 1000-fold or 2000-fold excess of unlabeled oligo with a 5′ extension, respectively. The unlabeled challenge oligo is the same as that used in reaction set C. (FIG. 7A) Single-strand oligonucleotides (51 nucleotides...