Efficient C2'alpha-hydroxylation of deoxyribose in protein-induced Z-form DNA.

Abstract

DNA local conformations are thought to play an important biological role in processes such as gene expression by altering DNA-protein interactions. Although left-handed Z-form DNA is one of the best-characterized and significant local structures of DNA, having been extensively investigated for more than two decades, the biological relevance of Z-form DNA remains unclear. This is presumably due to the lack of a versatile detection method in a living cell. Previously, we demonstrated that the incorporation of a methyl group at the guanine C8 position (m(8)G) dramatically stabilizes the Z-form of short oligonucleotides in a variety of sequences. To develop a photochemical method to detect Z-form DNA, we examined the photoreaction of 5-iodouracil-containing Z-form d(CGCG(I)UGCG)(ODN 1)/d(Cm(8)GCAm(8)GCG)(ODN 2) in 2 M NaCl and found stereospecific C2'alpha-hydroxylation occurred at G(4) to provide d(CGCrGUGCG), 5. Recently, Rich and co-workers [Schwartz et al. Science 1999, 284, 1841. Schwartz et al. Nat. Struct. Biol. 2001, 8, 761] found that an ubiquitous RNA editing enzyme, adenosine deaminase 1 (ADAR1), and tumor-associated protein DML-1 specifically bind to Z-form DNA. In the present study, we investigate the photoreactivity of octanucleotide ODN 1-2 in Z-form induced by Zalpha, which is the NH(2)-terminal domain of ADAR1 responsible for tight binding of ADAR1. Detailed product analysis revealed that the C2'alpha-hydroxylated products 5 and 6 produced significantly higher yields in Z-form ODN 1-2 induced by Zalpha compared with that in 2 M NaCl. Upon treatment with ribonuclease T1, 5 and 6 were quantitatively hydrolyzed at the 3'-phosphodiester bond of the rG residue to provide d(UGCG) as a common hydrolyzed fragment on the 3' side. Quantitative analysis demonstrated that the amount of photochemically formed 5 and 6 from ODN 1-2 directly correlated with the proportion of Z-form induced by Zalpha or NaCl. These results suggest that this photochemical and enzymatic procedure can be used as a specific probe for the existence of local Z-form structure in cellular DNA.

• Authors: Takanori Oyoshi, Kiyohiko Kawai, Hiroshi Sugiyama
• Journal: Journal of the American Chemical Society (J Am Chem Soc) Vol. 125 Issue 6 Pg. 1526-31 (Feb 12 2003) ISSN: 0002-7863 [Print] United States
• PMID: 12568612 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA-Binding Proteins
• Oligonucleotides
• RNA-Binding Proteins
• Deoxyribose
• Uracil
• DNA
• Ribonuclease T1
• ADARB1 protein, human
• Adenosine Deaminase
• 5-iodouracil

Topics

• Adenosine Deaminase (chemistry, metabolism)
• Chromatography, High Pressure Liquid
• Circular Dichroism
• DNA (chemistry, metabolism)
• DNA-Binding Proteins (chemistry, metabolism)
• Deoxyribose (chemistry, metabolism)
• Humans
• Hydroxylation
• Models, Molecular
• Nucleic Acid Conformation
• Oligonucleotides (chemical synthesis, chemistry)
• Photochemistry
• Protein Structure, Tertiary
• RNA-Binding Proteins
• Ribonuclease T1 (chemistry, metabolism)
• Stereoisomerism
• Substrate Specificity
• Uracil (analogs & derivatives, chemistry)