In keeping with the findings with the yeast Dna2△405N mutant, deletion of the Dna2 α1 helix reduced cleavage of the Rpa-coated 5'SL24 by a factor of ~2 compared to intact Dna2.
While the Col-r/r mutation renders the triple helix resistant to cleavage by MMPs, rodent MMP13 recognizes an additional cleavage site C-terminal to the N-telopeptide crosslink (Krane et al., 1996).
Therefore, we examined whether destabilisation of a substrate TM helix improves its cleavage by RseP MRE β-loop mutants.
Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer.
Each helical junction appears to undergo Mg2+-dependent coaxial stacking of its two arms, and it is believed that helix I, where the cleavage event occurs, folds into a cleft between helices II and VI.
However, replacement of the same residue by a helix-forming Trp residue resulted in no increase in cleavage, suggesting that helix destabilisation is important for improving substrate cleavage.
Transition charge values for cleavage sites between helix regions (31 41, 62 64) are similar to values for adjacent helix ends, indicating that helix separation does not precede helix unraveling.
This observation explains the relaxed need for helix-destabilizing residues for cleavage in detergent, because these TM segments are already partly unwound.
These data indicate that with the exception of Rnt1p, most RNase IIIs require a duplex longer than one turn of a helix to support intermolecular RNA cleavage.
Our study shows that a leucine at position 28 can substitute glutamine as Q28L variant showed similar stability, helix unwinding activity and dsRNA cleavage activity as that of HPR.
