Activation of DNA Cleavage by Oligomerization of DNA-Bound SgrAI

Abstract

SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of DNA activity and sequence specificity. Precleaved primary site DNA has been shown to be an allosteric effector [Hingorani-Varma & Bitinaite, (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR|CCGGYG, | indicates cut site, R=A,G, Y=C,T) and secondary (CR|CCGGY(A/C/T) and CR|CCGGGG) site DNA sequences. The fact that DNA is the allosteric effector of this endonuclease suggests at least two DNA binding sites on the functional SgrAI molecule, yet crystal structures of SgrAI [Dunten, et al., (2008) Nucleic Acids Res. 36, 5405–5416] show only one DNA duplex bound to one dimer of SgrAI. We show that SgrAI forms species larger than dimers or tetramers (High Molecular Weight Species, HMWS) in the presence of sufficient concentrations of SgrAI and its primary site DNA sequence, that are dependent on the concentration of the DNA bound SgrAI dimer. Analytical ultracentrifugation indicates that the HMWS is heterogeneous, has sedimentation coefficients of 15–20 s, and is composed of possibly 4–12 DNA bound SgrAI dimers. SgrAI bound to secondary site DNA will not form HMWS itself, but can bind to HMWS formed with primary site DNA and SgrAI. Uncleaved, as well as precleaved, primary site DNA is capable of stimulating HMWS formation. Stimulation of DNA cleavage by SgrAI, at primary as well as secondary sites, is also dependent on the concentration of primary site DNA (cleaved or uncleaved) bound SgrAI dimers. SgrAI bound to secondary site DNA does not have significant stimulatory activity. We propose that the oligomers of DNA bound SgrAI (i.e. HMWS) are the activated, or activatable, form of the enzyme.

Zinc enhances adiponectin oligomerization to octadecamers but decreases the rate of disulfide bond formation

“…Reactions with and without zinc were kept separate. After 2 h at 25°C, the oligomerization states of the reactions were assessed in a Beckman Coulter XL-I analytical ultracentrifuge as previously described (Park et al 2010) except absorbance scans were taken at 280 nm. The buffers loaded into the solvent compartment of the two-sector sedimentation velocity cells were aliquots of the dialysis buffer that were in contact with oligomerization reactions last.…”

Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization

“…SgrAI is one such “rare-cutting” endonuclease, which recognizes 3 degenerate primary (or canonical) octanucleotide sequences that differ in the 2 nd and 7 th bp – CGCCGGCG, CACCGGCG/CGCCGGTG, and CACCGGTG (Tautz et al, 1990). However, unlike any other REase, SgrAI will additionally cleave any of 14 secondary (or non-canonical) sequences (CPuCCGGPy(A/T/C) and CPuCCGGGG), but only in the presence of an activating primary site DNA containing a sufficient number of flanking bp (Bitinaite and Schildkraut, 2002; Park et al, 2010b; Wood et al, 2005). Thus, depending upon the input signal, the SgrAI REase can turn a rare DNA recognition sequence into one that is much more frequently encountered, dramatically increasing the number of DNA cleavages.…”

“…SgrAI maintains a baseline rate of cleavage for both primary and secondary sites, albeit one that is lower than its homologs (Bilcock et al, 1999; Park et al, 2010b). However, in the presence of multiple DNA cleavage sites, SgrAI acquires several properties that make the enzyme unique among known REases.…”

“…First, it relaxes the requirement for highly defined nucleotide recognition sites, enabling the cleavage of a total of 17 degenerate DNA sequences through a process known as sequence-specificity expansion (Bitinaite and Schildkraut, 2002). Second, the rate of subsequent DNA cleavage for primary and secondary sites is accelerated by up to several orders of magnitude (Hingorani-Varma and Bitinaite, 2003; Park et al, 2010b). Simultaneous sequence-specificity expansion and DNA cleavage rate acceleration have been more generally known as SgrAI activation (Park et al, 2010b).…”

“…Second, the rate of subsequent DNA cleavage for primary and secondary sites is accelerated by up to several orders of magnitude (Hingorani-Varma and Bitinaite, 2003; Park et al, 2010b). Simultaneous sequence-specificity expansion and DNA cleavage rate acceleration have been more generally known as SgrAI activation (Park et al, 2010b). In order for activation to occur, DNA containing the primary cleavage site must be present (Bitinaite and Schildkraut, 2002).…”