Impact of Cytosine 5-Halogens on the Interaction of DNA with Restriction Endonucleases and Methyltransferase

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 A 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.

Cleavage of Functionalized DNA Containing 5‐Modified Pyrimidines by Type II Restriction Endonucleases

“…. Denaturing PAGE analysis of 32 P-labeled template strands after PEX (lanes 2,4,6,8,11, and 13) and after PEX followed by cleavage with AfeI restriction endonuclease (lanes 3, 5, 7, 9, 12, and 14).…”

“…Marchionni et al [5] studied cleavage of DNA from bacteriophage l containing 5-bromodeoxyuridine with EcoRI, HindIII, and SmaI REs and concluded that the modified DNA was cleaved at the same site as the unmodified DNA. A series of DNAs containing some 5-halogenated and methylated deoxycytidines in the target sequence for MspI and HpaII REs were prepared by Valinluck et al, [6] who found that the cleavage of fluorinated DNA was slower, whereas the presence of the larger halogens Cl, Br, and I or of the methyl group at C completely prevented any cleavage. In order to verify the possible use of the REs in manipulation of pyrimidine-modified DNA bearing useful functional groups at position 5 and to study the influence of major-groove modifications on pyrimidines on the recognition of the DNA by REs, we have prepared model DNA sequences containing 5-ethynyl, 5-phenyl, and 5-(3-nitrophenyl) groups at U or C and studied their cleavage by diverse REs.…”

DNA methylation analysis of cancer-related genes in oral epithelial cells of healthy smokers

“…However, it has been suggested that oxidative stress can lead to the loss or gain of methylation. 76,77 Cigarette smoke contains reactive oxidants, which can cause macromolecular damage in exposed cells. Cigarette smoking also elicits a marked activation of leukocytes and platelets, which can also contribute to oxidative stress.…”

Intrinsic mutagenic properties of 5-chlorocytosine: A mechanistic connection between chronic inflammation and cancer

“…So far, research on 5ClC has focused on the lesion's ability to interact with DNA methyl transferases (22)(23)(24) when 5ClC is part of a CpG island; this interaction was shown to lead to changes in DNA methylation patterns (25). However, there are no reports affirming the ability of 5ClC to induce genetic changes directly during the replication or repair of genomic DNA.…”