MBP-Mcm10. Mcm10, with a Maltose Binding Protein (MBP) tag at the N-terminus, was purified by applying clarified extract from 12L E. coli overexpression cells at 1 ml/min to a 12 ml amylose column (New England Biolabs) in MBP buffer (20 mM Tris-Cl pH 7.5, 0.5 M NaCl, 1 mM DTT and 1 mM EDTA). The column was washed with 100 ml MBP buffer and eluted with 60 ml MBP buffer supplemented with 10 mM D-(+)-maltose (Sigma Aldrich) collecting 1.5 ml fractions. Peak fractions containing MBP-Mcm10 were pooled (~20 ml/85 mg) and dialyzed 2 hr vs. 2 L Buffer S (25 mM Hepes-KOH pH 7.5, 10% glycerol, 2 mM DTT, 2 mM MgCl2, and 1 mM EDTA). The dialysate was spun at 5,000 rpm in a swinging bucket rotor in an RC3B centrifuge to remove any precipitated material. The supernatant was applied to a 10 ml SP-Sepharose column that had been previously equilibrated in Buffer S supplemented with 100 mM NaCl and washed with 50 ml of the same buffer. The column was eluted with a 100 ml linear gradient from 100 to 600 mM NaCl in Buffer S. Peak fractions containing full-length MBP-Mcm10 were dialyzed overnight vs. 2L buffer S/200 mM NaCl and re-applied to a second 10 ml SP-Sepharose column and eluted with a step gradient from 20% to 50% of the buffer in pump B in 5% increments (pump A contained buffer S/100 mM NaCl and pump B contained buffer S/600 mM NaCl). The peak of full-length MBP-Mcm10 eluted at 50% pump B and was pooled and dialyzed against buffer S/200 mM NaCl in the presence of PreScission Protease to remove the MBP tag. Dialyzed pools were aliquoted, flash frozen in liquid nitrogen and stored at −80°C.