Impact of cytosine 5-halogens on the interaction of DNA with restriction endonucleases and methyltransferase

Victoria Valinluck, Winnie Wu, Pingfang Liu, Jonathan W. Neidigh, Lawrence Sowers

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine- containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 Å from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.

Original languageEnglish (US)
Pages (from-to)556-562
Number of pages7
JournalChemical Research in Toxicology
Volume19
Issue number4
DOIs
StatePublished - Apr 2006
Externally publishedYes

Fingerprint

Halogens
Cytosine
DNA Restriction Enzymes
Methyltransferases
Oligonucleotides
Flucytosine
Methylation
DNA
Halogenation
Deoxyribonuclease HpaII
Fluorine
DNA-Binding Proteins
Eukaryota
Electronic properties
Suicide
Carrier Proteins
Proteins
Substitution reactions
Inflammation
Molecules

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Impact of cytosine 5-halogens on the interaction of DNA with restriction endonucleases and methyltransferase. / Valinluck, Victoria; Wu, Winnie; Liu, Pingfang; Neidigh, Jonathan W.; Sowers, Lawrence.

In: Chemical Research in Toxicology, Vol. 19, No. 4, 04.2006, p. 556-562.

Research output: Contribution to journalArticle

Valinluck, Victoria ; Wu, Winnie ; Liu, Pingfang ; Neidigh, Jonathan W. ; Sowers, Lawrence. / Impact of cytosine 5-halogens on the interaction of DNA with restriction endonucleases and methyltransferase. In: Chemical Research in Toxicology. 2006 ; Vol. 19, No. 4. pp. 556-562.
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abstract = "Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine- containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 {\AA} from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.",
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AU - Sowers, Lawrence

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AB - Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine- containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 Å from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.

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