Chemical Nucleases: New Reagents in Molecular Biology

Sigman, David S.; Chen, Chi‐hong B.

doi:10.1146/annurev.bi.59.070190.001231

Cited by 137 publications

(67 citation statements)

References 33 publications

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“…The approach, based on using a chemical nuclease such as 1,10-phenantroline-copper (38), even permitted us to determine the approximate number of base pairs, the secondary structure of which had been perturbed by the cross-link. Importantly, this chemical nuclease does not represent measures of base pair disruption, as they can proceed even if the base pairing is distorted in a nondenaturational manner (40). The analysis of the duplex containing the site-specific interstrand cross-link of cisplatin has revealed that the interstrand crosslink of cisplatin results in distortions between ϳ4 to 5 base pairs at the d(GC)⅐d(GC) site involved in the cross-link (38).…”

Section: Discussionmentioning

confidence: 99%

Thermal and Thermodynamic Properties of Duplex DNA Containing Site-specific Interstrand Cross-link of Antitumor Cisplatin or Its Clinically Ineffective Trans Isomer

Hofr¹,

Brabec

2001

Journal of Biological Chemistry

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The effect of the single, site-specific interstrand crosslink formed by cisplatin or transplatin on the thermal stability and energetics of a 20-base pair DNA duplex is reported. The cross-linked or unplatinated 20-base pair duplexes were investigated with the aid of differential scanning calorimetry, temperature-dependent UV absorption, and circular dichroism. The cross-link of both platinum isomers increases the thermal stability of the modified duplexes by changing the molecularity of denaturation. The structural perturbation resulting from the interstrand cross-link of cisplatin increases entropy of the duplex and in this way entropically stabilizes the duplex. This entropic cross-link-induced stabilization of the duplex is partially but not completely compensated by the enthalpic destabilization of the duplex. The net result of these enthalpic and entropic effects is that the structural perturbation resulting from the formation of the interstrand cross-link by cisplatin induces a decrease in duplex thermodynamic stability, with this destabilization being enthalpic in origin. By contrast, the interstrand cross-link of transplatin is enthalpically almost neutral with the cross-link-induced destabilization entirely entropic in origin. These differences are consistent with distinct conformational distortions induced by the interstrand cross-links of the two isomers. Importantly, for the duplex cross-linked by cisplatin relative to that cross-linked by transplatin, the compensating enthalpic and entropic effects almost completely offset the difference in cross-link-induced energetic destabilization. It has been proposed that the results of the present work further support the view that the impact of the interstrand cross-links of cisplatin and transplatin on DNA is different for each and might also be associated with the distinctly different antitumor effects of these platinum compounds.The thermal and thermodynamic stability of DNA play an important role in many biological processes. In addition, agents of biological significance that modify DNA may also affect its thermal and thermodynamic stability, which may be associated with the mechanism underlying biological activity of such agents. Thus, the studies of thermal and thermodynamic stability of DNA modified by various agents are of great interest.It is well established that platinum coordination complexes exhibit antitumor effects (1-4). The success of platinum complexes in killing tumor cells results from their ability to form on DNA various types of covalent adducts that are capable of terminating DNA synthesis (5, 6) and the cellular processes triggered by the presence of those adducts on DNA (7). The first platinum complex introduced in the clinic is cis-diamminedichloroplatinum(II) (cisplatin) 1 (1). Although the antitumor effects of cisplatin were discovered more than 30 years ago, the mechanism of its antitumor activity has not yet been fully understood. It has been shown (8, 9) that this bifunctional platinum complex mainly forms intrastrand cross-l...

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Section: Discussionmentioning

confidence: 99%

Thermal and Thermodynamic Properties of Duplex DNA Containing Site-specific Interstrand Cross-link of Antitumor Cisplatin or Its Clinically Ineffective Trans Isomer

Hofr¹,

Brabec

2001

Journal of Biological Chemistry

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“…1). These single-cysteine variants provide unique sites for modification with either an azidophenacyl (AzP) group for photocrosslinking studies or acetamido-1,10-o-phenanthroline (AOP) for footprinting studies to identify nucleotides near P protein amino acids (Burgin and Pace 1990;Sigman and Chen 1990). Both of these reagents are proposed to react with RNA via direct contact (Burgin and Pace 1990;Sigman and Chen 1990) and thereby identify nucleotides within a 10-15 Å radius of the unique cysteine.…”

Section: Construction and Characterization Of Single-cysteine P Protementioning

confidence: 99%

“…To overcome this limitation, we applied directed hydroxylradical cleavage to map the regions of PRNA near P protein residues (Sigman and Chen 1990;Huber 1993). For these experiments, we used single-cysteine P protein mutants derivatized with acetamido-1,10-o-phenanthroline (AOP), which upon addition of copper results in a covalently attached copper(II)-phenanthroline (Cu-OP) moiety.…”

Section: Mapping the P Protein-prna Interfacementioning

confidence: 99%

Probing the architecture of the B. subtilis RNase P holoenzyme active site by cross-linking and affinity cleavage

Niranjanakumari¹,

Day-Storms²,

Ahmed³

et al. 2007

RNA

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Bacterial ribonuclease P (RNase P) is a ribonucleoprotein complex composed of one catalytic RNA (PRNA) and one protein subunit (P protein) that together catalyze the 59 maturation of precursor tRNA. High-resolution X-ray crystal structures of the individual P protein and PRNA components from several species have been determined, and structural models of the RNase P holoenzyme have been proposed. However, holoenzyme models have been limited by a lack of distance constraints between P protein and PRNA in the holoenzyme-substrate complex. Here, we report the results of extensive cross-linking and affinity cleavage experiments using single-cysteine P protein variants derivatized with either azidophenacyl bromide or 5-iodoacetamido-1,10-o-phenanthroline to determine distance constraints and to model the Bacillus subtilis holoenzyme-substrate complex. These data indicate that the evolutionarily conserved RNR motif of P protein is located near (<15 Å ) the pre-tRNA cleavage site, the base of the pre-tRNA acceptor stem and helix P4 of PRNA, the putative active site of the enzyme. In addition, the metal binding loop and N-terminal region of the P protein are proximal to the P3 stem-loop of PRNA. Studies using heterologous holoenzymes composed of covalently modified B. subtilis P protein and Escherichia coli M1 RNA indicate that P protein binds similarly to both RNAs. Together, these data indicate that P protein is positioned close to the RNase P active site and may play a role in organizing the RNase P active site.

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“…Copper ion is normally not included in OP derivatization, but is shown here to illustrate the Cu* to C, H distance. thoroughly studied for the free 2:1 1,10-phenanthroiinecopper complex (Sigman and Chen, 1990). All the products of the scission chemistry are known.…”

Section: Op Derivatizatjqqmentioning

confidence: 99%

“…The chemicai nuciease 1,10-phenanthroline-capper is a redox-active co-ordination compiex which cleaves the phosphodiester bond of nucleic acids by oxidative attack on the ribose or deoxyribose moiety (Sigman, 1990). Sequence-specific cieavage can be achieved if the chemical nuciease is tethered to a iigand with high affinity and seiectivity for a chosen DNA-recognition sequence (Sigman et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

DNA‐binding proteins as site‐specific nucleases

Pan¹,

Landgraf²,

Sigman³

1994

Molecular Microbiology

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SummaryDNA-binding proteins can be converted into sitespecific nucleases by linking them to the chemical nuclease 1,10-phenanthroline-copper. This can be readily accomplished by converting a minor grooveproximal amino acid to a cysteine residue using sitedirected mutagenesis and then chemically modifying the sulphydryl group with 5-iodoacetamido-1,10-phenanthroline-copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomai DNA, to test predictions based on high-resoiution nuclear magnetic resonance and X-ray crystal structures, and to locate binding sites of proteins within genomes.

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Chemical Nucleases: New Reagents in Molecular Biology

Cited by 137 publications

References 33 publications

Thermal and Thermodynamic Properties of Duplex DNA Containing Site-specific Interstrand Cross-link of Antitumor Cisplatin or Its Clinically Ineffective Trans Isomer

Thermal and Thermodynamic Properties of Duplex DNA Containing Site-specific Interstrand Cross-link of Antitumor Cisplatin or Its Clinically Ineffective Trans Isomer

Probing the architecture of the B. subtilis RNase P holoenzyme active site by cross-linking and affinity cleavage

DNA‐binding proteins as site‐specific nucleases

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