Monarch miniprep kits

Table 1 outlines the plasmids used in this study. Plasmids were propagated using NEB-10-beta competent cells (NEB) and purified using Monarch miniprep kits (NEB). Bacmids were generated using the Multibac system [27 (link)] and purified using alkaline lysis method followed by isopropanol precipitation and resuspension in TE buffer.

Table 1 outlines the plasmids and bacmids used in this study and their derivation. Plasmids were propagated using NEB-10-beta competent cells and purified using Monarch miniprep kits (NEB). Bacmids were generated using the Multibac system (Berger et al., 2004 (link)) and purified using alkaline lysis method followed by isopropanol precipitation and resuspension in TE.
Human FANCI:FANCD2 complex and Avi-ubiquitin was purified as described in Tan et al. (2020) . Xenopus laevis (frog) FANCI:FANCD2, human FANCB:FANCL:FAAP100, FANCC:FANCE:FANCF and UBE2T were expressed and purified as described in van Twest et al. (2017) (link). USP1:UAF1, HA-ubiquitin and UBE1 were purchased from Boston Biochem. ATR-ATRIP was purchased from Eurofins DiscoverX. Lambda phosphatase was purchased from New England Biolabs.

The catalytic DPPY motifs of both M.BceJIII and M.EcoGIX (Supplementary Figure S1) were converted into catalysis-defective mutant variants by introduction of an APPA substitution mutation using reverse PCR. Amplicons were obtained using plasmid templates carrying the wild type MTase allele. Primers P4 and P5 (M.BceJIII) and P10 and P11 (M.EcoGIX) introduced the mutations. The wild type plasmid templates in the amplification reaction were destroyed by DpnI digestion of the Dam-modified (G(m6A)TC) modified backbone to enrich for mutant variants.
Plasmids from candidate colonies were purified using Monarch miniprep kits (NEB, MA) and analyzed by sequencing and restriction digestion. Correct plasmids were identified, and retransformed into strain ER2796, which is deficient in all resident MTases as well as restriction systems (41 (link)), selecting Amp^r or Cm^r. Plasmid preparations from this background allowed us to use the RSII Pacific Biosciences sequencing instrument for modification detection and motif deduction.