Fig 1: NDRG1-MRE11 interaction is likely indirect. (A) Rosetta (DE3) competent cells were transformed with pET-28a-NDRG1 plasmids to express His-tagged full length WT NDRG1. Soluble extract was incubated with Ni-NTA beads and his-tagged NDRG1 proteins were eluted. Eluted proteins were concentrated then loaded onto Superdex75 10/300 column for size exclusion chromatography. Elution fractions containing desired NDRG1 were collected, run on SDS-PAGE, and stained with Coomassie Brilliant Blue R-250. Lane 1 (E1): most pure fraction, lane 2 (E2): less pure fractions pooled. (B) Sf21 insect cells were used to generate WT MRN complex: FLAG-Mre11-WT, Nbs1, 6xHis-Rad50. Baculovirus for each individual member of MRN complex were generated and Sf21 cells were infected with all three baculoviruses to generate full MRN complex. Soluble extract from insect cells containing MRN complex was incubated with Ni-NTA beads and his-tagged MRN complex was eluted. Eluted MRN complex was concentrated and loaded onto Superose 6 column for size exclusion chromatography. Elution fractions containing desired MRN complex were collected, run on SDS-PAGE, and stained with Coomassie Brilliant Blue R-250. lane 1 (E1): most pure fraction, lane 2 (E2): less pure fraction. (C) In vitro binding assay using purified FLAG-MRN complex and purified His-NDRG1 in the presence of various DNA substrates including double stranded DNA (dsDNA) 90 bp, herring testes (HT) DNA sheared, HT-DNA unsheared. FLAG M2 resin was used to pulldown FLAG-MRN complex, and eluate was subjected to western blotting with His tag antibody. (D) In vitro pulldown with purified FLAG-MRN complex and purified His-NDRG1 after SGK1 in vitro phosphorylation of His-NDRG1. (Left) In vitro phosphorylation reaction of his-NDRG1 in the absence and presence of ATP. (Right) In vitro pulldown using products from in vitro phosphorylation reaction. Black dots indicate the presence of specified proteins/compounds. (E) In vitro pulldown with purified FLAG-MRN complex and purified His-NDRG1 after SGK1 in vitro phosphorylation of His-NDRG1. (Left) In vitro phosphorylation reaction of his-NDRG1 in the absence and presence of SGK1. (Right) In vitro pulldown using products from in vitro phosphorylation reaction. (F) AlphaFold does not predict high confidence direct binding between MRE11-NDRG1. AlphaFold2 predictions of the best NDRG1-MRE11 multimer model. Top: Colored by chain (green = MRE11, cyan = NDRG1), Bottom: Colored by pLDDT (blue = high confidence, red = low confidence). Right: Zoomed out, Left: Zoomed in. (G) Predicted aligned error from AlphaFold prediction of NDRG1-MRE11 multimer. (H) Semi-endogenous pulldown using SUIT2 lysate with in vitro purified FLAG-MRN complex. SUIT2 cells were either untreated or treated with 2 mM hydroxyurea overnight. Lysates from SUIT2 cells were incubated with in vitro purified FLAG-MRN complex and M2 Flag resin overnight and probed for NDRG1 and MRE11 by Western blotting. (I) Semi-endogenous pulldown using SUIT2 lysate with in vitro purified FLAG-MRN complex. SUIT2 cells were either untreated or treated with lambda phosphatase before incubating with the purified FLAG-MRN complex and M2 Flag resin overnight, and probed for phospho- and tNDRG1 by Western blotting. (J) Semi-endogenous pull-down using Ni-NTA beads that were pre-conjugated to in vitro purified His-NDRG1, and Ni-NTA-His-NDRG1 beads were added to HEK293T cell lysates expressing GFP-MRE11, after which the beads were probed for the presence of GFP-MRE11. The uncropped blots are shown in Supplementary Materials.