DNA Distortion and Specificity in a Sequence-Specific Endonuclease

Abstract

SummaryFive new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca 2+ , Mg 2+ , and Mn 2+ ) are presented. While previous structures were produced from soaking Ca 2+ into pre-formed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca 2+ , Mg 2+ , or Mn 2+ . The Mn 2+ bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca 2+ allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA bound enzyme, and show how DNA conformational energetics determine DNA cleavage rates by the Q138F HincII enzyme.Many fundamentally important functions within cells are mediated by proteins which recognize and bind to specific DNA sequences. Studies of sequence specific DNA binding proteins indicate that specific target sequences are recognized using both direct and indirect readout 1 . Direct readout involves direct contacts such as hydrogen bonds and van der Waals interactions between the protein and the DNA base functional groups, mostly in the major groove. Indirect readout is defined as sequence specificity occurring in the absence of such direct contacts, and has been proposed to involve contacts mediated by water 2 or other small molecules 3 , as well as distortions of DNA that can distinguish different sequences energetically 4-13 (Little, et al., submitted for publication).Restriction endonucleases are a class of enzymes found in bacteria which cleave DNA at specific target sequences and are thought to protect the bacterial cell from phage infection 14 . The target sites in the host DNA are methylated by a methyltransferase, and thereby protected from the cleavage activity of the endonuclease. Cleavage at sequences other than the target site can result in damage and possibly death to the bacterial cell, as only the target site sequence is protected by the activity of the cognate methyltransferase enzyme. Therefore, restriction *Corresponding author, NCH: Telephone: 520-626-3828 FAX: 520-626-9288 nhorton@u.arizona.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCESSION NUMBERSCoordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 3E3Y, 3E40, 3E41, 3E42, 3E43, 3E44, 3E45. NIH Public...

Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity

“…This results in this study are similar to previous examinations of the mechanism of indirect readout for restriction endonucleases such as EcoRV (Martin et al, 1999) and HincII (Babic et al, 2008; Joshi et al, 2006). In those studies, the effect of substituted bases or protein side chains was more deleterious toward DNA cleavage than binding.…”

Early Interrogation and Recognition of DNA Sequence by Indirect Readout

“…1C. The numbering of nucleotides in the crystal structure for the same base pairs is 5-10, which has been used in previous reports (Babic, 2008; Horton et al, 2002; Joshi et al, 2006)). Removal of the N141 side chain by mutation to alanine removes the direct readout of these two bases, leaving any residual specificity to be performed solely by indirect readout.…”

Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease

“…In most cases, the crystal structures are solved with one version of the degenerate target site and only for HincII, the structures were obtained with two cognate oligonucleotide variants providing us with the first structural mechanism of the alternative sequence recognition. Crystallographic studies supported by biochemical experiments ( 12 , 13 ) demonstrate that HincII specificity for the degenerate GTYRAC sequence arises from the indirect readout of the conformational preferences at the central pyrimidine–purine step ( 8 ). However, it remains to be established whether the same or different mechanisms are responsible for the degenerate sequence recognition by other restriction enzymes.…”