Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

Abstract

Nucleoside/nucleotide analogs that lack the 3′-hydroxy group are widely utilized for HIV therapy. These chain-terminating nucleoside analogs (CTNAs) block DNA synthesis after their incorporation into growing DNA, leading to the antiviral effects. However, they are also considered to be DNA damaging agents, and tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme, is reportedly able to remove such CTNA-modifications of DNA. Here, we have synthesized phosphoramidite building blocks of representative CTNAs, such as acyclovir, abacavir, carbovir, and lamivudine, and oligonucleotides with the 3′-CTNAs were successfully synthesized on solid supports. Using the chemically synthesized oligonucleotides, we investigated the excision of the 3′-CTNAs in DNA by the human excision repair cross complementing protein 1-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease, which is one of the main components of the nucleotide excision repair pathway. A biochemical analysis demonstrated that the ERCC1-XPF endonuclease cleaved 2–7 nt upstream from the 3′-blocking CTNAs, and that DNA synthesis by the Klenow fragment was resumed after the removal of the CTNAs, suggesting that ERCC1-XPF participates in the repair of the CTNA-induced DNA damage.

Keywords

• DNA damage
• DNA repair
• antiviral agents
• solid-phase synthesis
• nucleotide excision repair