Indirect Readout of DNA Sequence by Proteins: The Roles of DNA Sequence‐Dependent Intrinsic and Extrinsic Forces

Koudelka, Gerald B.; Mauro, Steven A.; Ciubotaru, Mihai

doi:10.1016/s0079-6603(06)81004-4

Cited by 45 publications

(37 citation statements)

References 123 publications

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“…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 water2 or other small molecules3, as well as distortions of DNA that can distinguish different sequences energetically 4–13(Little, et al, submitted for publication ).…”

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confidence: 99%

DNA Distortion and Specificity in a Sequence-Specific Endonuclease

Babić

Little

Manohar

et al. 2008

Journal of Molecular Biology

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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...

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confidence: 99%

DNA Distortion and Specificity in a Sequence-Specific Endonuclease

Babić

Little

Manohar

et al. 2008

Journal of Molecular Biology

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“…Thus, the effects of base sequence changes on affinity may even be smaller than we reported. If this were the case, that could suggest that 933W repressor discriminates between these various DNA sites solely by recognizing sequence-dependent differences in DNA structure [42], a mechanism known as indirect readout [35], [43]. Indeed we suggest that 933W repressor discriminates between operators bearing various sequences at positions 7, 8 and 7′ by indirect readout.…”

Section: Resultsmentioning

confidence: 81%

Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

et al. 2012

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We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein.

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“…This type of indirect readout may be strongly influenced by local, sequence-dependent DNA conformation (38,39). These sequence-dependent effects may be further amplified by DNA substrate deformation in this region of the I-PpoI substrate complex, and thus interfere with correct positioning of the scissile phosphate (Figures 1 and 4) (21,26).…”

Section: Discussionmentioning

confidence: 99%

Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease

Eklund¹,

Ulge²,

Eastberg³

et al. 2007

Nucleic Acids Research

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We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or ‘homing’ site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA–protein interface. Biochemical characterization of these proteins revealed a wide range of site–binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.

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Indirect Readout of DNA Sequence by Proteins: The Roles of DNA Sequence‐Dependent Intrinsic and Extrinsic Forces

Cited by 45 publications

References 123 publications

DNA Distortion and Specificity in a Sequence-Specific Endonuclease

DNA Distortion and Specificity in a Sequence-Specific Endonuclease

Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease

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