Control of Hexamerization, Assembly, and Excluded Strand Specificity for the Sulfolobus solfataricus MCM Helicase

Abstract

A growing body of evidence supports a steric exclusion and wrapping model for DNA unwinding in which hexameric helicases interact with the excluded single-stranded DNA (ssDNA) in addition to the encircled strand. Interactions with the excluded ssDNA have been shown to be mediated primarily by electrostatic interactions, but base stacking with surface-exposed tyrosine residues is an alternative hypothesis. Here, we mutated several external tyrosine and positively charged residues from full-length Sulfolobus solfataricus MCM along the proposed path of excluded strand binding and assessed their impact on DNA unwinding. Four of the five tyrosine residues had significant decreases in their level of unwinding, and one, Y519A, located within the α/β-α linker region of the C-terminal domain, had the most severe perturbation attributed to the disruption of hexamerization. The Y519 mutant exhibits an enhanced and stabilized secondary structure that is modulated by temperature, binding DNA with a higher apparent affinity and suggesting a pathway for hexameric assembly. Hydrogen/deuterium exchange coupled to mass spectrometry was used to map deuterium uptake differences between wild-type and Y519A apo structures highlighting global differences in solvent accessible areas consistent with altered quaternary structure. Two of the five electrostatic mutants had significantly reduced levels of DNA unwinding and combined with previous mutations better define the exterior binding path. The importance of the electrostatic excluded strand interaction was confirmed by use of morpholino DNA substrates that showed analogous reduced unwinding rates. These results better define the hexameric assembly and influence of the excluded strand interactions in controlling DNA unwinding by the archaeal MCM complex.

Amidst multiple binding orientations on fork DNA, Saccharolobus MCM helicase proceeds N-first for unwinding

“…Previously, multiple reports have shown that the fraction unwound by Sso MCM generally hovers between 0.3 and 0.5 depending on the substrate and conditions (Barry et al, 2007; Graham et al, 2011; Graham et al, 2018). The proportion of Sso MCM bound in a productive orientation and state can be determined in a single-turnover DNA unwinding experiment.…”

Mechanisms of hexameric helicases

“…Interestingly, cryo-EM structures have shown that dsDNA can enter far into the N-terminal region of CMG before it splits into separated strands and that loops in this region seem to divert the displaced strand to a possible exit tunnel between Mcm3 and Mcm5 (Yuan et al 2020). As with SF-IV helicases, it has been suggested that the exterior surface of MCM helicases may associate with the excluded strand, an interaction that has been proposed to prevent backsliding (Rothenberg et al 2007;Costa et al 2008;Graham et al 2011Graham et al , 2016Graham et al , 2018. However, structural studies of eukaryotic replisome components suggest that the Pol a-primase complex sits close to the N-terminal (fork-proximal) tier of CMG through an interaction with the Ctf4/AND-1 scaffolding protein (Zhu et al 2007;Simon et al 2014;Sun et al 2015b;Georgescu et al 2017;Yuan et al 2019).…”