Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3'-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease.
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.
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan. yamamoto@chem.es.osaka-u.ac.jp.
Junpei
J
Yamamoto
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan. takahata@bio.chem.es.osaka-u.ac.jp.
Chiaki
C
Takahata
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan. kuraoka@chem.es.osaka-u.ac.jp.
Isao
I
Kuraoka
Department of Chemistry, Tokyo Metropolitan University, 1-1 Minami-Ohsawa, Hachi-Ohji, Tokyo 192-0397, Japan. khirota@tmu.ac.jp.
Kouji
K
Hirota
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan. iwai@chem.es.osaka-u.ac.jp.
Shigenori
S
Iwai
D000212:Acyclovir / Q000627:therapeutic use; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D006678:HIV / Q000235:genetics* / Q000472:pathogenicity; D015658:HIV Infections / Q000188:drug therapy / Q000235:genetics*; D006801:Humans; D009705:Nucleosides / Q000235:genetics* / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000627:therapeutic use
D004268:DNA-Binding Proteins; D009705:Nucleosides; D009943:Organophosphorus Compounds; C434331:phosphoramidite; C095888:xeroderma pigmentosum group F protein; C071447:ERCC1 protein, human; D004720:Endonucleases; D000212:Acyclovir
DNA damage; DNA repair; antiviral agents; nucleotide excision repair; solid-phase synthesis