Characterization of BcgI, a new kind of restriction-modification system.

Kong, Huimin; Roemer, Stefan; Waite-Rees, Phyllis A.; Benner, Jack S.; Wilson, Geoffrey G.; Nwankwo, Donald O.

doi:10.1016/s0021-9258(17)42403-3

Cited by 55 publications

(10 citation statements)

References 39 publications

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“…DNA binding dissociation constants for Bam HI endonuclease are comparable (). Table S1 (Supporting Information) features these examples and additional details for these and a number of other systems ( 18 , 20 , − .…”

Section: Discussionmentioning

confidence: 99%

Dissecting the Metal Ion Dependence of DNA Binding by PvuII Endonuclease

Conlan

Dupureur

2002

Biochemistry

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Divalent cations can provide an effective means of modulating the behavior of nucleic acid binding proteins. As a result, there is strong interest in understanding the role of metal ions in the function of both nucleic acid binding proteins and their enzymes. We have applied complementary fluorescence spectroscopic and nitrocellulose filter binding assays to quantitate the role of metal ions in mediating DNA binding and sequence specificity by the representative PvuII endonuclease. At pH 7.5 in the presence of the catalytically nonsupportive Ca(II), this enzyme binds the PvuII target sequence with a K(d) of 50 pM. Under strict metal-free conditions, the enzyme exhibits a K(d) of only 300 nM for the cognate sequence, an affinity which is weak relative to those measured for other systems in the absence of metal ions. This represents a 6000-fold increase in PvuII affinity for cognate DNA upon the addition of Ca(II). The pH dependences of both metal ion-dependent and metal ion-independent DNA binding are remarkably shallow throughout the physiological range; other characterized restriction enzymes exhibit more pronounced pH dependences of DNA binding even in the absence of metal ions. Similar measurements with noncognate sequences indicate that divalent metal ions are not important to nonspecific DNA binding; K(d) values are approximately equal to 200 nM throughout the physiological pH range, a behavior shared with other endonucleases. While some of these results extend somewhat the range of expected behavior for restriction enzymes, these results indicate that PvuII endonuclease shares with other characterized systems a mechanism by which cognate affinity and sequence discrimination are most effectively achieved in the presence of divalent metal ions.

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Section: Discussionmentioning

confidence: 99%

Dissecting the Metal Ion Dependence of DNA Binding by PvuII Endonuclease

Conlan

Dupureur

2002

Biochemistry

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“…Although many restriction endonucleases form stable complexes with DNA in the absence of Mg 2+ , they do not necessarily bind specifically. For example, EcoRV, TaqI, Cfr9I, and BcgI bind equally well to DNA containing or lacking their recognition sites (Taylor et al, 1991;Zebala et al, 1992;Siksnys & Pleckaityte, 1993;Kong et al, 1994). In this respect, I-PpoI is similar to the restriction endonucleases EcoRI, BamHI, RsrI, FokI, and SmaI (Halford & Johnson, 1980;Terry et al, 1983;Aiken et al, 1991;Xu & Schildkraut, 1991b;Waugh & Sauer, 1993;Withers & Dunbar, 1995), which bind to their recognition sites specifically in the absence of a divalent metal ion.…”

Section: Comparison Of Catalysis By I-ppoi and Restriction Endonucleasesmentioning

confidence: 99%

Substrate Binding and Turnover by the Highly Specific I-PpoI Endonuclease

Wittmayer

Raines

1996

Biochemistry

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Intron-encoded endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with high specificity. I-PpoI endonuclease, an intron-encoded endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) that catalyzes the cleavage of a large asymmetric DNA sequence (15 base pairs). Here, the interactions of I-PpoI with its substrate were examined during both binding (in the absence of Mg2+) and catalysis (in the presence of Mg2+). Using circular permutation assays, I-PpoI was shown to bend its substrate by 38 +/- 4 degrees upon binding. Two independent methods, gel mobility shift assays and fluorescence polarization assays, revealed that I-PpoI binds tightly to its substrate. Values of Kd range from 3.3 to 112 nM, increasing with increasing NaCl concentration. Similar salt effects on the values of Km were observed during steady-state catalysis. At low salt concentrations, the value of kcat/Km for the cleavage of an oligonucleotide duplex approaches 10(8) M-1 s-1. Although other divalent cations can replace Mg2+, catalysis by I-PpoI is most efficient in the presence of an oxophilic metal ion that prefers an octahedral geometry: Mg2+ > Mn2+ > Ca2+ = Co2+ > Ni2+ > Zn2+. Together, these results provide the first chemical insight into substrate binding and turnover by an intron-encoded endonuclease.

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“…In the presence of Ca 2+ , a nonreactive analogue of Mg 2+ that does not allow catalysis, R.EcoRV shows considerable preference for GATATC sites (9,12). Studies have placed R.BamHI (13), 1 R.RsrI (14), and R.SmaI (15) in the R.EcoRI family and R.TaqI (16), R.Cfr9I (17), R.Cfr10I (18), R.BcgI (19), R.MunI (20,21), and R.BglI (22) with R.EcoRV.…”

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confidence: 99%

Binding and Recognition of GATATC Target Sequences by the EcoRV Restriction Endonuclease: A Study Using Fluorescent Oligonucleotides and Fluorescence Polarization

et al. 2001

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Oligonucleotides labeled with hexachlorofluorescein (hex) have enabled the interaction of the restriction endonuclease EcoRV with DNA to be evaluated using fluorescence anisotropy. The sensitivity of hex allowed measurements at oligonucleotide concentrations as low as 1 nM, enabling K(D) values in the low nanomolar range to be measured. Both direct titration, i.e., addition of increasing amounts of the endonuclease to hex-labeled oligonucleotides, and displacement titration, i.e., addition of unlabeled oligonucleotide to preformed hex-oligonucleotide/EcoRV endonuclease complexes, have been used for K(D) determination. Displacement titration is the method of choice; artifacts due to any direct interaction of the enzyme with the dye are eliminated, and higher fluorescent-labeled oligonucleotide concentrations may be used, improving signal-to-noise ratio. Using this approach (with three different oligonucleotides) we found that the EcoRV restriction endonuclease showed a preference of between 1.5 and 6.5 for its GATATC target sequence at pH 7.5 and 100 mM NaCl, when the divalent cation Ca2+ is absent. As expected, both the presence of Ca2+ and a decrease in pH value stimulated the binding of specific sequences but had much less effect on nonspecific ones.

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Characterization of BcgI, a new kind of restriction-modification system.

Cited by 55 publications

References 39 publications

Dissecting the Metal Ion Dependence of DNA Binding by PvuII Endonuclease

Dissecting the Metal Ion Dependence of DNA Binding by PvuII Endonuclease

Substrate Binding and Turnover by the Highly Specific I-PpoI Endonuclease

Binding and Recognition of GATATC Target Sequences by the EcoRV Restriction Endonuclease: A Study Using Fluorescent Oligonucleotides and Fluorescence Polarization

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