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高分辨率补骨脂素图谱揭示了SV40调控区域中DNA螺旋结构的改变。

High resolution psoralen mapping reveals an altered DNA helical structure in the SV40 regulatory region.

作者信息

Ostrander E A, Karty R A, Hallick L M

机构信息

Department of Microbiology and Immunology, Oregon Health Sciences University, Portland 97201.

出版信息

Nucleic Acids Res. 1988 Jan 11;16(1):213-27. doi: 10.1093/nar/16.1.213.

Abstract

Preferential psoralen photobinding sites have been mapped in vitro on restriction fragments spanning the SV40 origin region and surrounding sequences by a new fine structure analysis technique. Purified DNA fragments were photoreacted with 3H-5-methylisopsoralen (3H-5-MIP), a psoralen derivative which forms only monoadducts. Fragments were then end-labeled and digested with lambda exonuclease, a 5' processive enzyme which we have determined pauses at 5-MIP monoadducts. When photobinding sites were mapped on denaturing sequencing gels, it was observed that 5-MIP binds preferentially to 5'-TA sites, and to a lesser degree to 5'-AT sites. Utilizing this approach, we have identified a psoralen hypersensitive region in which the binding sites were much stronger than those in the surrounding sequences. This region extends from 150 base pairs (bp) to the late side of the enhancers to the early enhancer/promoter boundary. We suggest that this region contains a sequence directed structural alteration of the DNA helix which can be detected by the psoralen mapping approach described.

摘要

通过一种新的精细结构分析技术,已在体外将补骨脂素光结合位点定位到跨越SV40起始区域及周边序列的限制性片段上。纯化的DNA片段与3H - 5 - 甲基异补骨脂素(3H - 5 - MIP)进行光反应,3H - 5 - MIP是一种仅形成单加合物的补骨脂素衍生物。然后对片段进行末端标记,并用λ外切核酸酶消化,λ外切核酸酶是一种5' 渐进性酶,我们已确定其在5 - MIP单加合物处会停顿。当在变性测序凝胶上定位光结合位点时,观察到5 - MIP优先结合到5'-TA位点,对5'-AT位点的结合程度较低。利用这种方法,我们确定了一个补骨脂素高敏区域,其中的结合位点比周边序列中的结合位点强得多。该区域从150个碱基对(bp)延伸至增强子的晚期一侧到早期增强子/启动子边界。我们认为该区域包含一种DNA螺旋的序列导向结构改变,可通过所述的补骨脂素定位方法检测到。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6296/334622/1e8324658342/nar00143-0226-a.jpg

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