xmni  (New England Biolabs)

Journal: bioRxiv

Article Title: Leading and lagging strand abasic sites differentially affect vertebrate replisome progression but involve analogous bypass mechanisms

doi: 10.1101/2025.01.09.632187

Figure Lengend Snippet: A) DNA oligonucleotides oMC30 and oMC31 were annealed to form a blunt duplex which was cloned into the PsiI restriction site of pJD161. This insert contains Nt.BbvCI restriction sites placed 63 nucleotide (nt) apart on the same strand. Incorporation of the oMC30/31 duplex in the forward direction generated pMC9, which allows for for incorporation of a modified oligonucleotide between the Nt.BbvCI sites on the Top strand. Incorporation of the oMC30/31 duplex in the reverse direction generated pMC10 and pMC14, which allow for incorporation of a modified on the Bottom strand. Replication forks can approach the modified region of pMC14 from either direction and thus encounter the modified strand on either the leading or lagging strand template . pMC9 and MC10 harbor a lacO array downstream of the indicated sequence to ensure the modified strand is encountered on the lagging (pMC9) or leading (pMC10) strand template when incubated in LacR to assemble a replication barrier. B) To insert a site- and strand-specific AP site into plasmid DNA, pMC14 was first nicked with Nt.BbvCI. A DNA oligonucleotide containing a site-specific dUracil was then annealed and ligated into the gap between nicking sites. Excess unligated oligos and nicked plasmid intermediates were then removed and the remaining fully ligated plasmid was purified. C) pMC14 was modified with a Uracil-containing oligonucleotide (pdUracil) or an unmodified control oligo (pCtrl), as in (B), then digested with XmnI and MscI to screen for incorporation of the dUracil. XmnI linearizes the plasmid into a 3 kb fragment to eliminate differences between plasmids that arise from different levels of supercoiling. MscI targets the same position as the inserted dUracil modification, which blocks digestion by MscI. Resistance to digestion by MscI acts as a read out for incorporation of the site-specific dUracil modification. pdUracil was resistant to MscI (compare lane 9 with lanes 3 and 6), which confirmed that dUracil was efficiently incorporated. D) pdUracil was treated with UDG to generate plasmids containing an AP site (pAP),then treated with APE1 to test for the generation of an AP site. APE1 converts AP sites to single-stranded DNA breaks. Thus, conversion of supercoiled plasmids to nicked species indicates the presence of AP sites. Parental plasmid (pMC14) was included as a marker for supercoiled (SC) DNA, and also nicked with Nt.BbvCI as a marker for nicked plasmid. Treatment with UDG and APE1 resulted in predominantly nicked plasmids (lane 6), which demonstrated efficient conversion of dUracil to AP sites. E) To evaluate the stability of pAP in Xenopus egg extracts, plasmid DNA containing an abasic site (pAP) was incubated in High Speed Supernatant (HSS) or buffer control (Egg Lysis Buffer, ELB) then treated with APE1 to assess the presence of the AP site. pAP became resistant to APE1 following incubation with HSS (compare lanes 2 and 4), which indicated that the AP site was repaired by Xenopus egg extracts. To test whether LacR binding adjacent to the AP site could confer stability in Xenopus egg extracts, pAP was pre-incubated with LacR before being incubated in HSS. Most plasmids became nicked under these conditions (compare lanes 5 and 7), which indicated that the LacR-bound AP site was incised in HSS but not fully repaired. Thus, LacR-binding conferred partial stability to the AP site in Xenopus egg extracts. F) Phosphorothioate-modified AP site plasmid (p*AP*) was incubated in Xenopus egg extracts with or without LacR to evaluate stability of the AP site compared to pAP, which lacked phosphorothioates. In addition to High Speed Supernatant (HSS) of Xenopus egg extracts, HSS plus NucleoPlasmic Extract (NPE)) combined (H+N) was also used to mimic the final extract concentration during a typical replication reaction using Xenopus egg extracts. pAP incubated with LacR became nicked (compare lanes 5-6 with lane 4), p*AP* did not (compare lanes 11-12 with lane 10). Thus, p*AP* incubated with LacR was stabilized in Xenopus egg extracts compared to pAP. G) To evaluate whether p*AP* retained the AP site in Xenopus egg extracts, Products from (F) were purified then digested with XmnI and MscI (as in (C)). The ∼3000 bp and ∼700 bp fragments of XmnI-MscI digestion (C) increased when pAP was incubated with LacR (compare lanes 5-6 with lane 4), but not when p*AP* was incubated with LacR (compare lanes 11-12 with lane 10). Note that a background level of MscI digest products is detected even incubation with Xenopus egg extracts (lanes 1, 4, 7, 10) because the AP site was not fully resistant to MscI, in contrast to dUracil (in (C)). Note also that presence of the AP site could not be evaluated by treatment with APE1 (as in (D)-(E)) because phosphorothioate modifications confer resistance to APE1. Overall, these data indicate that the combination of phosphorothioates and LacR binding stabilize AP sites in Xenopus egg extracts. H) To quantitatively assess AP site retention in Xenopus egg extracts, retention of the ∼3000 bp product from (G) was quantified. Values were normalized to the ELB condition to account for partial sensitivity of the AP site to MscI. The combination of phosphorothioates and LacR (p*AP*+LacR) resulted in no appreciable loss of AP sites. This demonstrated that AP sites could be stabilized in Xenopus egg extracts.

Article Snippet: To validate modified plasmids, 300 ng of plasmids were digested with 5 units of XmnI with or without 5 units of MscI in 1x rCutsmart buffer (New England Biolabs) in a reaction volume of 10 μl at 37°C.

Techniques: Clone Assay, Generated, Modification, Sequencing, Incubation, Plasmid Preparation, Purification, Control, Marker, Lysis, Binding Assay, Concentration Assay

Journal: eLife

Article Title: Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy

doi: 10.7554/eLife.97438

Figure Lengend Snippet: Activation of MCM-micrococcal nuclease (MNase) in G1-arrested wild-type (WT) (16747), sir2 (16769), sir2fun30 (17257), and fun30 (17256) cells at the rARS with calcium will eliminate the 3.5 kb XmnI fragment (upper panel). PIK1 , a single-copy gene in which we detect no MCM binding, is used as a loading control. Normalized ARS1200 band intensity at 15 m is expressed relative to time 0. Quantitation of the uncut band was used to infer relative ribosomal DNA (rDNA) array size in sir2 fun30 mutant at 0.35 relative to WT. Figure 3—source data 1. PDF file containing original Southern blots displayed in indicating the relevant bands, treatments and band sizes. Figure 3—source data 2. Original files for Southern blot analysis displayed in . Figure 3—source data 3. Quantification of bands in Southern blots and calculations of MCM loading displayed in .

Article Snippet: DNA was extracted from spheroplasts using the YeaStar Genomic DNA Kit (Zymo Research), digested with XmnI, separated on 1.75% agarose gels and analyzed by standard Southern blotting technique.

Techniques: Activation Assay, Binding Assay, Control, Quantitation Assay, Mutagenesis, Southern Blot