Hoang Trung M, Kent Tatiana, Pomerantz Richard T
Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA.
Bio Protoc. 2017 Jun 20;7(12). doi: 10.21769/BioProtoc.2330.
DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3' termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human Polθ is able to modify the 3' terminal ends of both DNA and RNA with a wide array of large and diverse ribonucleotide and deoxyribonucleotide analogs, its terminal transferase activity is more useful for biotechnology applications than terminal deoxynucleotidyl transferase (TdT). Here, we present in detail simple methods by which purified human Polθ is utilized to modify the 3' terminal ends of RNA and DNA for various applications in biotechnology and biomedical research.
DNA聚合酶θ（Polθ）是一种杂乱的酶，对于易错的DNA双链断裂（DSB）修复途径——替代末端连接（alt-EJ）至关重要。在这种形式的DSB修复过程中，Polθ通过模板化和非模板化的核苷酸添加循环在切除的DSB的3'末端进行末端转移酶活性。由于人类Polθ能够用多种大的和不同的核糖核苷酸和脱氧核糖核苷酸类似物修饰DNA和RNA的3'末端，其末端转移酶活性在生物技术应用中比末端脱氧核苷酸转移酶（TdT）更有用。在这里，我们详细介绍了利用纯化的人类Polθ修饰RNA和DNA的3'末端以用于生物技术和生物医学研究中的各种应用的简单方法。
