fpg  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

doi: 10.1093/nar/gkv116

Figure Lengend Snippet: R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.

Article Snippet: For controls, 20 units of Endo III (New England Biolabs) and 0.2 pmol (10 nM) a double-stranded substrate containing an AP site and a 5′-32 P label on the top strand was incubated in 10 mM Tris–HCl (pH 7.5), 100 mM NaCl and 1 mM EDTA (total 20 μl) at 37°C for 20 min followed by 100 mM NaBH4 (or 100 mM NaCl) as above.

Techniques: Incubation, Concentration Assay, SDS Page, Polyacrylamide Gel Electrophoresis

Journal: PLoS ONE

Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

doi: 10.1371/journal.pone.0157270

Figure Lengend Snippet: TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

Article Snippet: As positive control, the oligo AP was incubated with 1U Endo III (E . coli NTH1, New England Biolabs), or with 2U Exonuclease III (Exo III, E . coli AP endonuclease, New England Biolabs) or with 1 μg of purified recombinant TcAP1 T . cruzi AP endonuclease.

Techniques: Activity Assay, Labeling, Incubation, Negative Control, Positive Control, Purification, Transformation Assay, Transfection

Journal: bioRxiv

Article Title: MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

doi: 10.1101/2020.02.13.947598

Figure Lengend Snippet: Extent of leaf variegation observed for different msh1 mutant alleles. A. An example of an msh1 mutant (CS3372) individual with a leaf-variegation phenotype. B. Values represent the percent of individuals in an F3 family from a homozygous mutant F2 parent that showed visible leaf variegation at time of harvest for mitochondrial and plastid DNA extraction. Means and standard errors are from three replicate F3 families from each mutant line (see Fig. 2 ). Between 45 and 66 individuals were scored for each family. Lowercase letters indicate significant differences between alleles based on a Tukey’s HSD test. Consistent with its lower rate of observed sequence and structural variation in cytoplasmic genomes, the SALK_046763 msh1 mutant line exhibited less severe phenotypic effects.

Article Snippet: Half of the cleaned sample was then treated with a cocktail of repair enzymes to remove single-stranded damage in a 50 μl reaction volume containing 1x NEB CutSmart Buffer, 8 U Fpg (New England Biolabs M0240), 5 U Uracil-DNA Glycosylase (New England Biolabs M0280), and 10 U Endonuclease III (New England Biolabs M0268) for 30 min at 37° C. Samples were then cleaned with 1.6 volumes of SPRI beads.

Techniques: Mutagenesis, DNA Extraction, Sequencing

Journal: bioRxiv

Article Title: MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

doi: 10.1101/2020.02.13.947598

Figure Lengend Snippet: Crossing design to test candidate nuclear genes involved in RRR of cytoplasmic genomes. Using a wild-type maternal plant (black) and either a homozygous mutant (red) or heterozygous pollen donor, we generated a heterozygous F1 individual that carried cytoplasmic genomes inherited from a wild-type lineage (as indicated by the black mitochondrion). After selfing the F1, we genotyped the resulting F2 progeny to identify three homozygous mutants and three homozygous wild-type individuals. Given that the mutations in candidate RRR genes are expected to be recessive, the F2 generation would be the first in which the sampled cytoplasmic genomes were exposed to the effects (red asterisks) of these mutants. The identified F2 individuals were each allowed to self-fertilize and set seed to produce multiple F3 families that all inherited their cytoplasmic genomes from the same F1 grandparent. The F3 families were used for purification of mitochondrial and plastid DNA for duplex sequencing. Sequencing was performed on three replicate families for each genotype. Arabidopsis silhouette image is from PhyloPic (Mason McNair).

Article Snippet: Half of the cleaned sample was then treated with a cocktail of repair enzymes to remove single-stranded damage in a 50 μl reaction volume containing 1x NEB CutSmart Buffer, 8 U Fpg (New England Biolabs M0240), 5 U Uracil-DNA Glycosylase (New England Biolabs M0280), and 10 U Endonuclease III (New England Biolabs M0268) for 30 min at 37° C. Samples were then cleaned with 1.6 volumes of SPRI beads.

Techniques: Mutagenesis, Generated, Purification, Sequencing

Journal: bioRxiv

Article Title: MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

doi: 10.1101/2020.02.13.947598

Figure Lengend Snippet: Intact MSH1 transcripts but reduced expression level in homozygous SALK_046763 msh1 mutants. A. Sanger trace from cDNA sequencing confirms that properly spliced transcripts are present in SALK_046763 msh1 mutants despite the large T-DNA insertion in intron 8. The vertical line below the trace indicates the location of the expected splice junction between exons 8 and 9. B. ΔC T values are calculated based on the difference in quantitative reverse-transcriptase PCR (qRT-PCR) threshold cycle value for each indicated MSH1 marker and the geometric mean of the threshold cycle values from two reference genes ( UBC and UBC9 ). Means and standard errors are from four biological replicates (F4 plants derived from crossing design described in Fig. 2 ), each of which is based on the mean of three technical replicates. The SALK_046763 mutants exhibit higher ΔC T (indicating lower MSH1 expression). Both MSH1 markers indicate a similar shift in ΔC T values (2.3 cycles for exons 8/9 and 2.5 cycles for exon 16), corresponding to an approximately 5-fold difference in transcript abundance. Significant differences between mutant and wild type genotypes at a level of P

Article Snippet: Half of the cleaned sample was then treated with a cocktail of repair enzymes to remove single-stranded damage in a 50 μl reaction volume containing 1x NEB CutSmart Buffer, 8 U Fpg (New England Biolabs M0240), 5 U Uracil-DNA Glycosylase (New England Biolabs M0280), and 10 U Endonuclease III (New England Biolabs M0268) for 30 min at 37° C. Samples were then cleaned with 1.6 volumes of SPRI beads.

Techniques: Expressing, Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Derivative Assay, Mutagenesis