Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin

Li Lan,Leizhen Wei,Luxi Sun,Marcel Bruchez,Akira Yasui,Arthur S Levine,Robert W Sobol,Satoshi Nakajima,Ching-Lung Hsieh,Bennett Van Houten

doi:10.1093/nar/gkt1233

Li Lan, Leizhen Wei + Show 8 more

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https://doi.org/10.1093/nar/gkt1233

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Abstract

Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Highlights

Reactive oxygen species (ROS) can be generated endogenously during cellular respiration or in response to infection and exogenously by chemical and physical agents
This plasmid was integrated into a genome site at $200 copies [20] and the chromatin status at the site of the integrated plasmid was clarified using a FISH assay to define the precise genomic site of tetR-KR or TA-KR binding in the U2OS/tetracycline response element (TRE) cell line
Staining with DAPI showed a region of relatively condensed DNA at this repeated array (Figure 1B, lower panel) as well as a highly condensed centromere, indicating that the TRE array exists in a heterochromatinized region

Summary

Introduction

Reactive oxygen species (ROS) can be generated endogenously during cellular respiration or in response to infection and exogenously by chemical and physical agents. ROS mainly induce oxidized bases and single-strand breaks (SSBs) in DNA. These lesions are repaired via the base excision/SSB repair (BER/SSBR) pathways [1,2]. XRCC1, a scaffold protein that accumulates at sites of SSBs in association with PAR, is necessary for repair progression as it recruits other repair factors [4]. Both BER and SSBR are carried out with short-patch or long-patch repair synthesis by DNA polymerases and completed with ligation by DNA ligase III or DNA ligase I. DNA polymerase ß (Polß), which contains an N-terminus dRP lyase domain and a C-terminal polymerase domain, is involved in both short- and long-patch BER [8,9]

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