Open AccessModification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity
Author(s) -
Trung Hoang,
Tatiana Kent,
Richard T. Pomerantz
Publication year - 2017
Publication title -
bio-protocol
Language(s) - English
Resource type - Journals
ISSN - 2331-8325
DOI - 10.21769/bioprotoc.2330
Subject(s) - terminal deoxynucleotidyl transferase , dna polymerase , deoxyribonucleotide , polymerase , microbiology and biotechnology , biology , dna , transferase , ribonucleotide , biochemistry , nucleotide , enzyme , oligonucleotide , gene , tunel assay , apoptosis
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.