Efficient C2'alpha-hydroxylation of deoxyribose in protein-induced Z-form DNA.
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.
Oyoshi
Takanori
T
Division of Biofunctional Molecules, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kanda, Chiyoda, Tokyo 101-0062, Japan.
Kawai
Kiyohiko
K
Sugiyama
Hiroshi
H
D000243:Adenosine Deaminase / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003855:Deoxyribose / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010777:Photochemistry; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D006163:Ribonuclease T1 / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D003855:Deoxyribose; D014498:Uracil; D004247:DNA; D006163:Ribonuclease T1; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase; C048747:5-iodouracil