DNA unwinding mechanism of a eukaryotic replicative CMG helicase

Abstract

DNAThe DNAs used for these studies were the following oligonucleotides (Integrated DNA Technologies) of: a 45-mer lagging-strand oligo, (5ʹ-GGCAGGCAGGCAGGCACACACTCTCCAATTA/iBiodT/CACTTCCTACTCTA-3ʹ) and a 70-mer leading-strand oligo (5ʹ-TAGAGTAGGAAGTGA/iBiodT/AATTGGAGAGTGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T-3ʹ). The asterisks are residues containing a phosphothio linkage. The two oligos were annealed in equimolar amounts by heating to 90°C followed by slow (1 hour) cooling to room temperature. The hybrid was purified from a 8% native PAGE then a 1 molar equivalent of streptavidin was added, enabling a single streptavidin to cross-link the two diametrically opposed biotins. ProteinsMcm10 and CMG were purified as previously described (Langston et al., 2017), with the following modifications. The Mcm10 contained a N-terminal hexahistidine tag and a C-terminal 3X FLAG tag. Briefly, 48 L E. coli cells carrying the Mcm10 T7 based E. coli expression vector were grown to OD 0.6 at 37 o C, then cooled to 15 o C and induced upon adding IPTG for an additional 8 h at 15 o C. The cells were harvested by slow speed centrifugation and lysed using a continuous flow high speed homogenizer. Cell debris was removed by centrifugation and applied to a 10 ml Chelating Sepharose Fast Flow column (GE Healthcare) charged with 50 mM NiSO 4 in Buffer A (20 mM Tris-Cl pH 7.9, 5 mM imidazole, 500 mM NaCl, 0.01% NP-40). The column was washed with Buffer A, then eluted with 375 mM imidazole in Buffer A. The eluated material was applied to a 6 ml anti-FLAG M2 affinity gel (Sigma) equilibrated in Buffer B (20 mM Tris-Cl pH 7.5, 10% glycerol, 500 mM NaCl, 1 mM DTT, 1 mM MgCl 2 , 0.01% NP-40) and then washed with 20 column volumes of Buffer B before eluting with Buffer B containing 0.2 mg/ml FLAG peptide (EZ Biolab, Carmel, Indiana USA) using two 6 ml pulses of 20 min each and collecting 1.5 ml fractions. Eluted fractions were analyzed by SDS-PAGE, protein concentration was determined using Bradford Protein Stain (Sigma) wtih BSA as a standard. Proteins were then aliquoted, snap frozen in liquid nitrogen and stored at -80 o C.The CMG-Mcm10 complex was reconstituted by mixing 765 pmol CMG with 3.1 nmol Mcm10 in 0.7 ml Buffer C (10 mM Tris-Cl pH 7.5, 200 mM KCl, 2mM DTT, 2 mM MgCl 2 ) for 30 minutes on ice. The mixture was then applied to a 0.1 ml MonoQ column, equilibrated in Buffer C. The column was washed using the same buffer and eluted with a 2.5 ml gradient of Buffer C from 0.2 M KCl to 0.6 M KCl. Fractions of 0.1 ml were collected, analysed by Bradford and SDS-PAGE, pooled and dialyzed against 50 mM K-glutamate in 25 mM Tris-acetate pH 7.5, 2 mM Mg acetate, and 1 mM DTT. Dialysed material was analyzed again by Bradford stain for protein concentation , then aliquoted, snap frozen in liquid nitrogen and stored at -80 o C.

CryoEM structures of human CMG–ATPγS–DNA and CMG–AND-1 complexes

“…In our structure, all six MCM subunits contact ssDNA, spanning a total of 11 nucleotides. The footprint of an MCM subunit on ssDNA covers four nucleotides, rather than two as previously reported ( 19 , 49 ), with two overlapping nucleotides between neighbouring subunits (Figure 3 ). We believe that the difference in footprint relative to previous reports for the yeast and Drosophila CMGs ( 19 , 49 ) is due to the improved resolution of our study.…”

“…The footprint of an MCM subunit on ssDNA covers four nucleotides, rather than two as previously reported ( 19 , 49 ), with two overlapping nucleotides between neighbouring subunits (Figure 3 ). We believe that the difference in footprint relative to previous reports for the yeast and Drosophila CMGs ( 19 , 49 ) is due to the improved resolution of our study. As the MCM C-tier amino acids at the protein-ssDNA interface are invariant in yeast and Drosophila MCMs ( Supplementary Figure S7 ), our description of the protein-DNA footprint is universally applicable.…”

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

“…In living cells, many more proteins associate with replisomes than are required for minimal biochemical reconstructions 9,33 . These interactions may be constitutive or induced by replication stress, but are typically interpreted as representing a single complex (see Introduction) 12,22,[34][35][36][37][38][39][40][41][42] .…”

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

“…In living cells many more proteins associate with replisomes than are required for “minimal” biochemical reconstructions 37,12 . These interactions may be constitutive or induced by replication stress, but are typically interpreted as representing a single complex (see Introduction) 15,22,38–42 .…”