Activation of DNA cleavage by oligomerization of DNA-bound SgrAI

Chad K. Park, Amanda P. Stiteler, Santosh Shah, M. Imran Ghare, Jurate Bitinaite, Nancy C Horton

Research output: Contribution to journalArticle

15 Citations (Scopus)

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, K., and Bitinaite, J. (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR|CCGGYG, where the vertical bar indicates a cut site, R denotes A or G, and Y denotes C or 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, P. W., 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, forms of the enzyme.

Original languageEnglish (US)
Pages (from-to)8818-8830
Number of pages13
JournalBiochemistry
Volume49
Issue number41
DOIs
StatePublished - Oct 19 2010

Fingerprint

Oligomerization
DNA Cleavage
Chemical activation
DNA
Molecular Weight
Dimers
Molecular weight
DNA sequences
Type II Site Specific Deoxyribonucleases
Endonucleases
Ultracentrifugation
Nucleic Acids
Sedimentation
Oligomers
Binding Sites

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

Cite this

Park, C. K., Stiteler, A. P., Shah, S., Ghare, M. I., Bitinaite, J., & Horton, N. C. (2010). Activation of DNA cleavage by oligomerization of DNA-bound SgrAI. Biochemistry, 49(41), 8818-8830. https://doi.org/10.1021/bi100557v

Activation of DNA cleavage by oligomerization of DNA-bound SgrAI. / Park, Chad K.; Stiteler, Amanda P.; Shah, Santosh; Ghare, M. Imran; Bitinaite, Jurate; Horton, Nancy C.

In: Biochemistry, Vol. 49, No. 41, 19.10.2010, p. 8818-8830.

Research output: Contribution to journalArticle

Park, CK, Stiteler, AP, Shah, S, Ghare, MI, Bitinaite, J & Horton, NC 2010, 'Activation of DNA cleavage by oligomerization of DNA-bound SgrAI', Biochemistry, vol. 49, no. 41, pp. 8818-8830. https://doi.org/10.1021/bi100557v
Park CK, Stiteler AP, Shah S, Ghare MI, Bitinaite J, Horton NC. Activation of DNA cleavage by oligomerization of DNA-bound SgrAI. Biochemistry. 2010 Oct 19;49(41):8818-8830. https://doi.org/10.1021/bi100557v
Park, Chad K. ; Stiteler, Amanda P. ; Shah, Santosh ; Ghare, M. Imran ; Bitinaite, Jurate ; Horton, Nancy C. / Activation of DNA cleavage by oligomerization of DNA-bound SgrAI. In: Biochemistry. 2010 ; Vol. 49, No. 41. pp. 8818-8830.
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