Generation of DNA single-strand displacement by compromised nucleotide excision repair.

Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylation (γH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients.

Kinetics of endogenous mouse FEN1 in base excision repair.

The structure specific flap endonuclease 1 (FEN1) plays an essential role in long-patch base excision repair (BER) and in DNA replication. We have generated a fluorescently tagged FEN1 expressing mouse which allows monitoring the localization and kinetics of FEN1 in response to DNA damage in living cells and tissues. The expression of FEN1, which is tagged at its C-terminal end with enhanced yellow fluorescent protein (FEN1-YFP), is under control of the endogenous Fen1 transcriptional regulatory elements. In line with its role in processing of Okazaki fragments during DNA replication, we found that FEN1-YFP expression is mainly observed in highly proliferating tissue. Moreover, the FEN1-YFP fusion protein allowed us to investigate repair kinetics in cells challenged with local and global DNA damage. In vivo multi-photon fluorescence microscopy demonstrates rapid localization of FEN1 to local laser-induced DNA damage sites in nuclei, providing evidence of a highly mobile protein that accumulates fast at DNA lesion sites with high turnover rate. Inhibition of poly (ADP-ribose) polymerase 1 (PARP1) disrupts FEN1 accumulation at sites of DNA damage, indicating that PARP1 is required for FEN1 recruitment to DNA repair intermediates in BER.

PARP inhibition versus PARP-1 silencing: different outcomes in terms of single-strand break repair and radiation susceptibility.

The consequences of PARP-1 disruption or inhibition on DNA single-strand break repair (SSBR) and radio-induced lethality were determined in synchronized, isogenic HeLa cells stably silenced or not for poly(ADP-ribose) polymerase-1 (PARP-1) (PARP-1(KD)) or XRCC1 (XRCC1(KD)). PARP-1 inhibition prevented XRCC1-YFP recruitment at sites of 405 nm laser micro irradiation, slowed SSBR 10-fold and triggered the accumulation of large persistent foci of GFP-PARP-1 and GFP-PCNA at photo damaged sites. These aggregates are presumed to hinder the recruitment of other effectors of the base excision repair (BER) pathway. PARP-1 silencing also prevented XRCC1-YFP recruitment but did not lengthen the lifetime of GFP-PCNA foci. Moreover, PARP-1(KD) and XRCC1(KD) cells in S phase completed SSBR as rapidly as controls, while SSBR was delayed in G1. Taken together, the data demonstrate that a PARP-1- and XRCC1-independent SSBR pathway operates when the short patch repair branch of the BER is deficient. Long patch repair is the likely mechanism, as GFP-PCNA recruitment at photo-damaged sites was normal in PARP-1(KD) cells. PARP-1 silencing elicited hyper-radiosensitivity, while radiosensitization by a PARP inhibitor reportedly occurs only in those cells treated in S phase. PARP-1 inhibition and deletion thus have different outcomes in terms of SSBR and radiosensitivity.

Radiosensitization by the poly(ADP-ribose) polymerase inhibitor 4-amino-1,8-naphthalimide is specific of the S phase of the cell cycle and involves arrest of DNA synthesis.

Radiosensitization caused by the poly(ADP-ribose) polymerase (PARP) inhibitor 4-amino-1,8-naphthalimide (ANI) was investigated in 10 asynchronously growing rodent (V79, CHO-Xrs6, CHO-K1, PARP-1+/+ 3T3, and PARP-1-/- 3T3) or human (HeLa, MRC5VI, IMR90, M059J, and M059K) cell lines, either repair proficient or defective in DNA-PK (CHO-Xrs6 and M059J) or PARP-1 (PARP-1-/- 3T3). Pulse exposure to ANI (1-hour contact) potentiated radiation response in rodent cells except in PARP-1(-/-) 3T3 fibroblasts. In contrast, ANI did not significantly enhance radiation susceptibility in asynchronously dividing human cells; yet, single-strand break rejoining was lengthened by ca. 7-fold in all but mouse PARP-1-/- 3T3s. Circumstantial evidence suggested that radiosensitization by ANI occurs in rapidly dividing cells only. Experiments using synchronized HeLa cells consistently showed that ANI-induced radiosensitization is specific of the S phase of the cell cycle and involves stalled replication forks. Under these conditions, prolonged contact with ANI ended in the formation of de novo DNA double-strand breaks hours after irradiation, evoking collision with uncontrolled replication forks of DNA lesions whose repair was impaired by inhibition of the PARP catalytic activity. The data suggest that increased response to radiotherapy by PARP inhibitors may be achieved only in rapidly growing tumors with a high S-phase content.