FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks.

Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.

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.

Repair synthesis step involving ERCC1-XPF participates in DNA repair of the Top1-DNA damage complex.

Topoisomerase 1 (Top1) is the intercellular target of camptothecins (CPTs). CPT blocks DNA religation in the Top1-DNA complex and induces Top1-attached nick DNA lesions. In this study, we demonstrate that excision repair cross complementing 1 protein-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease and replication protein A (RPA) participate in the repair of Top1-attached nick DNA lesions together with other nucleotide excision repair (NER) factors. ERCC1-XPF shows nuclease activity in the presence of RPA on a 3'-phosphotyrosyl bond nick-containing DNA (Tyr-nick DNA) substrate, which mimics a Top1-attached nick DNA lesion. In addition, ERCC1-XPF and RPA form a DNA/protein complex on the nick DNA substrate in vitro, and co-localize in CPT-treated cells in vivo. Moreover, the DNA repair synthesis of Tyr-nick DNA lesions occurred in the presence of NER factors, including ERCC1-XPF, RPA, DNA polymerase delta, flap endonuclease 1 and DNA ligase 1. Therefore, some of the NER repair machinery might be an alternative repair pathway for Top1-attached nick DNA lesions. Clinically, these data provide insights into the potential of ERCC1 as a biomarker during CPT regimens.

Xeroderma pigmentosum group F protein binds to Eg5 and is required for proper mitosis: implications for XP-F and XFE.

The xeroderma pigmentosum group F-cross-complementing rodent repair deficiency group 1 (XPF-ERCC1) complex is a structure-specific endonuclease involved in nucleotide excision repair (NER) and interstrand cross-link (ICL) repair. Patients with XPF mutations may suffer from two forms of xeroderma pigmentosum (XP): XP-F patients show mild photosensitivity and proneness to skin cancer but rarely show any neurological abnormalities, whereas XFE patients display symptoms of severe XP symptoms, growth retardation and accelerated aging. Xpf knockout mice display accelerated aging and die before weaning. These results suggest that the XPF-ERCC1 complex has additional functions besides NER and ICL repair and is essential for development and growth. In this study, we show a partial colocalization of XPF with mitotic spindles and Eg5. XPF knockdown in cells led to an increase in the frequency of abnormal nuclear morphology and mitosis. Similarly, the frequency of abnormal nuclei and mitosis was increased in XP-F and XFE cells. In addition, we showed that Eg5 enhances the action of XPF-ERCC1 nuclease activity. Taken together, these results suggest that the interaction between XPF and Eg5 plays a role in mitosis and DNA repair and offer new insights into the pathogenesis of XP-F and XFE.

Strand breakage of a (6-4) photoproduct-containing DNA at neutral pH and its repair by the ERCC1-XPF protein complex.

The (6-4) photoproduct is one of the major UV-induced lesions in DNA. We previously showed that hydrolytic ring opening of the 5' base and subsequent hydrolysis of the glycosidic bond of the 3' component occurred when this photoproduct was treated with aqueous NaOH. In this study, we found that another product was obtained when the (6-4) photoproduct was heated at 90 °C for 6 h, in a 0.1 M solution of N,N'-dimethyl-1,2-ethanediamine adjusted to pH 7.4 with acetic acid. An analysis of the chemical structure of this product revealed that the 5' base was intact, whereas the glycosidic bond at the 3' component was hydrolyzed in the same manner. The strand break was detected for a 30-mer oligonucleotide containing the (6-4) photoproduct upon treatment with the above solution or other pH 7.4 solutions containing biogenic amines, such as spermidine and spermine. In the case of spermidine, the rate constant was calculated to be 1.4 × 10(-8) s(-1) at 37 °C. The strand break occurred even when the oligonucleotide was heated at 90 °C in 0.1 M sodium phosphate (pH 7.0), although this treatment produced several types of 5' fragments. The Dewar valence isomer was inert to this reaction. The product obtained from the (6-4) photoproduct-containing 30-mer was used to investigate the enzymatic processing of the 3' end bearing the damaged base and a phosphate. The ERCC1-XPF complex removed several nucleotides containing the damaged base, in the presence of replication protein A.