Structure of Bam HI Endonuclease Bound to DNA: Partial Folding and Unfolding on DNA Binding

Newman, Matthew; Strzelecka, Teresa E.; Dorner, Lydia F.; Schildkraut, Ira; Aggarwal, Aneel K.

doi:10.1126/science.7624794

Cited by 312 publications

(265 citation statements)

References 34 publications

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“…The structures of the five type II endonucleases determined by x-ray diffraction, EcoRI (1), BamHI (2,3), EcoRV (4,5), PvuII (6,7) and Cfr10I (8), share substantial elements of similarity. Analysis of the enzymes complexed to DNA, where known, suggests a preliminary classification into two different groups (9).…”

mentioning

confidence: 99%

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Nastri

Evans

Walker

et al. 1997

Journal of Biological Chemistry

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The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5 -CAGCTG-3 ) through the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA-or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.The structures of the five type II endonucleases determined by x-ray diffraction, EcoRI (1), BamHI (2, 3), EcoRV (4, 5), PvuII (6, 7) and Cfr10I (8), share substantial elements of similarity. Analysis of the enzymes complexed to DNA, where known, suggests a preliminary classification into two different groups (9). The endonucleases that produce 5Ј-overhanging ends, EcoRI and BamHI, approach the DNA from the major groove, where most of their base-specific contacts are made. The endonucleases that produce blunt-ended DNA products, EcoRV and PvuII, approach the DNA from the minor groove side and establish contacts in the major groove by wrapping around the DNA. Structure-function studies have been limited to a small number of type II enzymes. Years of studies have produced an extensive amount of information about EcoRI (10 -18), EcoRV (18 -24), and NaeI (25) and, more recently, BamHI (26,27). These studies have focused on the identification and analysis of residues involved in catalysis, testing alternative mechanisms of catalysis, and understanding how a specific DNA sequence is recognized. Attempts to alter the sequence specificity have proven to be difficult, with successful examples limited to mutants showing relaxed specificity (10,11,28) or displaying activity toward DNA substituted with unnatural bases (15,29). One explanation for this difficulty is that a change in specificity not only requires recognition of a different DNA sequence, but also requires that the linkage between recognition and catalysis be retained. A class of mutations that might illuminate this linkage is the catalytic mutations, where specific DNA binding is retained but catalysis is reduced (26, 30). In particular, mutants in this class that are outside the catalytic center might be deficient in the linkage between recognition and ca...

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confidence: 99%

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Nastri

Evans

Walker

et al. 1997

Journal of Biological Chemistry

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“…In our structure, we observed that the inferred active site was more visible than in the structure of Dyda et al (15); however, we found that a surface loop (residues 141-148) was still disordered. In many wellstudied enzymes, flexible surface loops are important for substrate binding and catalysis (27)(28)(29), leading us to focus on the role of the region of residues 140-149. We substituted Gly residues at 140 and 149, potential hinges for the integrase flexible loop, with more constrained Ala residues and examined the mutant structures and enzymatic activities.…”

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confidence: 99%

The Mobility of an HIV-1 Integrase Active Site Loop Is Correlated with Catalytic Activity^,

et al. 1999

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Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 Å resolution. The structure extends helix R4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of R4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop is important for a postbinding catalytic step.Integration of the viral cDNA into a host chromosome is required for viral replication. Prior to integration, viral cDNA is cleaved on each strand near the 3′ end by integrase (terminal cleavage), probably to remove nontemplated extra bases occasionally added by reverse transcriptase (1, 2). Integrase then catalyzes the attachment of the recessed 3′ ends to the target DNA (strand transfer) (3-5). In vitro, purified integrase protein can carry out the terminal cleavage (6, 7) and strand transfer reactions (8-11). Purified integrase can also carry out an apparent reversal of strand transfer, termed "disintegration" (12).The structure of full-length integrase has not yet been identified. However, the structure of each of its three domains has been determined in isolation. The amino-terminal domain (amino acids 1-50) is composed of three R-helices with an embedded zinc binding site (13,14). The central domain [amino acids 50-212 (50-212 1 )] is composed of mixed R-helix and -sheet (15), forming a compact fold seen previously in several polynucleotidyl phosphotransferases (16)(17)(18)(19). This domain by itself can carry out covalent chemistry on permissive disintegration substrates, an indication of its role in catalysis (20,21). A conserved amino acid sequence motif, D,D-35-E, is found in the central domain of integrases and some related prokaryotic transposases. The carboxyl-terminal domain (amino acids 213-288) is composed of a five-stranded -barrel resembling an SH3 domain, and contributes to DNA binding (22,23). All three domains form dimers independently, and the full-length integrase is likely to act as a higher-order multimer (reviewed in ref 24).We have used X-ray crystallography and protein engineering to investigate the active site of HIV-1 integrase....

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“…The recent cocrystal structure of BamHI with a cognate oligodeoxynucleotide [7] is compatible with both mechanisms. As already described the catalytic centre is formed by three acidic residues because Glu-ll3 replaces the lysine residue found in the three other enzymes.…”

Section: Eeorimentioning

confidence: 68%

“…On the other hand, the known three dimensional structures of four restriction enzymes, namely EcoRI [2], EcoRV, PvulI [4,5], and BamHI [6,7], show striking homologies. The overall folds of EcoRI and BamHI as well as parts of the folds of EcoRV and PvulI are similar [8].…”

Section: Introductionmentioning

confidence: 99%

Asp‐59 is not important for the catalytic activity of the restriction endonuclease EcoRI

1996

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Structure of Bam HI Endonuclease Bound to DNA: Partial Folding and Unfolding on DNA Binding

Cited by 312 publications

References 34 publications

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

The Mobility of an HIV-1 Integrase Active Site Loop Is Correlated with Catalytic Activity^,

Asp‐59 is not important for the catalytic activity of the restriction endonuclease EcoRI

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Structure of Bam HI Endonuclease Bound to DNA: Partial Folding and Unfolding on DNA Binding

Cited by 312 publications

References 34 publications

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

The Mobility of an HIV-1 Integrase Active Site Loop Is Correlated with Catalytic Activity,

Asp‐59 is not important for the catalytic activity of the restriction endonuclease EcoRI

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The Mobility of an HIV-1 Integrase Active Site Loop Is Correlated with Catalytic Activity^,