| Autor: |
Graham BW; Department of Chemistry , University of Pittsburgh , Pittsburgh , Pennsylvania 15260 , United States., Bougoulias ME; Department of Chemistry and Biochemistry , Baylor University , Waco , Texas 76798 , United States., Dodge KL; Department of Chemistry and Biochemistry , Baylor University , Waco , Texas 76798 , United States., Thaxton CT; Department of Chemistry and Biochemistry , Baylor University , Waco , Texas 76798 , United States., Olaso D; Department of Chemistry and Biochemistry , Baylor University , Waco , Texas 76798 , United States., Tao Y; Department of Chemistry , Florida State University , Tallahassee , Florida 32306 , United States., Young NL; Verna & Marrs McLean Department of Biochemistry & Molecular Biology , Baylor College of Medicine , One Baylor Plaza , Houston , Texas 77030-3411 , United States., Marshall AG; Department of Chemistry , Florida State University , Tallahassee , Florida 32306 , United States.; National High Magnetic Field Laboratory , 1800 East Paul Dirac Drive , Tallahassee , Florida 32310 , United States., Trakselis MA; Department of Chemistry and Biochemistry , Baylor University , Waco , Texas 76798 , United States. |
| Abstrakt: |
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. |
