Importance of phosphate contacts for sequence recognition by EcoRI restriction enzyme.

Abstract:

:We have studied the importance of charge and hydrogen-bonding potential of the phosphodiester backbone for binding and cleavage by EcoRI restriction endonuclease. We used 12-mer oligodeoxynucleotide substrates with single substitutions of phosphates by chiral methylphosphonates at each position of the recognition sequence -pGpApApTpTpCp-. Binding was moderately reduced between 4- and 400-fold more or less equally for the R(P) and S(P)-analogues mainly caused by missing charge interaction. The range of cleavage effects was much wider. Four substrates were not cleaved at all. At both flanking positions and in the purine half of the sequence up to the central position, cleavage was more impaired than binding and differences between R(P) and S(P) diastereomeres were more pronounced. These effects are easily interpreted by direct phosphate contacts seen in the crystal structure. For the effects of substitutions in the pyrimidine half of the recognition sequence, more indirect effects have to be discussed.

authors

Rosati O,Srivastava TK,Katti SB,Alves J

doi

10.1016/s0006-291x(02)00645-9

subject

Has Abstract

pub_date

2002-07-05 00:00:00

pages

198-205

issue

1

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(02)00645-9

journal_volume

295

pub_type

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