Christensen Laura A, Wang Hong, Van Houten Bennett, Vasquez Karen M
Department of Carcinogenesis, Science Park-Research Division, University of Texas MD Anderson Cancer Center, Smithville, TX, USA.
Nucleic Acids Res. 2008 Dec;36(22):7136-45. doi: 10.1093/nar/gkn880. Epub 2008 Nov 7.
Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.
光反应性补骨脂素可在双链DNA中形成链间交联（ICL）。在真细菌中，核酸内切酶UvrABC在处理补骨脂素ICL中起关键作用。补骨脂素修饰的三链形成寡核苷酸（TFO）可用于将ICL引导至特定基因组位点。先前对富含嘧啶的甲氧补骨脂素修饰的TFO的研究表明，该TFO可抑制UvrABC的切割作用。由于不同的化学性质可能会改变TFO引导的ICL的处理过程，因此我们研究了由富含嘌呤的TFO形成的另一种三链体对UvrABC核酸酶处理4'-（羟甲基）-4,5',8-三甲基补骨脂素（HMT）ICL的影响。使用HMT修饰的TFO将ICL引导至特定位点，我们发现UvrABC在双链体富含嘌呤的链上，在ICL的3'侧约3个碱基处以及5'侧约9个碱基处、TFO结合区域内进行切割。与先前的报道相反，UvrABC核酸酶切割TFO引导的补骨脂素ICL的效率高于单独的补骨脂素ICL。此外，TFO被UvrA和UvrB从其双链体结合位点解离。由于TFO引导的ICL诱变需要核苷酸切除修复，这些损伤的有效处理支持使用三链体技术引导DNA损伤进行基因组修饰。
