In situ analysis of repair processes for oxidative DNA damage in mammalian cells

Abstract

Oxidative DNA damage causes blocks and errors in transcription and replication, leading to cell death and genomic instability. Although repair mechanisms of the damage have been extensively analyzed in vitro, the actual in vivo repair processes remain largely unknown. Here, by irradiation with an UVA laser through a microscope lens, we have conditionally produced single-strand breaks and oxidative base damage at restricted nuclear regions of mammalian cells. We showed, in real time after irradiation by using antibodies and GFP-tagged proteins, rapid and ordered DNA repair processes of oxidative DNA damage in human cells. Furthermore, we characterized repair pathways by using repair-defective mammalian cells and found that DNA polymerase ␤ accumulated at single-strand breaks and oxidative base damage by means of its 31-and 8-kDa domains, respectively, and that XRCC1 is essential for both polymerase ␤-dependent and proliferating cell nuclear antigen-dependent repair pathways of single-strand breaks. Thus, the repair of oxidative DNA damage is based on temporal and functional interactions among various proteins operating at the site of DNA damage in living cells.

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

“…While PARP1 depletion does not affect PCNA recruitment, as shown previously [34,36,53], it decreases PARG recruitment to DNA damage sites [36,37]. Using simultaneous dual-channel imaging we showed that in the absence of PARP1, PARG and PCNA are recruited with a comparable kinetic mechanism at a single cell level.…”

“…Both laser settings induced fast and transient recruitment of PARP1, coupled with transient accumulation of PAR, in contrast to sustained accumulation of γH2AX, which persisted > 30 min after irradiation (Figure 1). While PARP1 recruitment and PAR production may be used as a marker of both SSBs and DSBs [29,34], phosphorylation of the histone variant H2AX (γH2AX) is primarily induced at DSBs and is known to spread megabases away from the damage site and persist beyond the time required for DSB repair [45,46]. Given that PAR and γH2AX do not exhibit the same distribution along the laser stripe (Figure 1, bottom overlay panels), both SSBs and DSBs seem to be formed under our experimental conditions.

10.1080/19491034.2018.1516485-F0001Figure 1. DNA damage induction with a UV-A 355 nm laser coupled with BrdU presensitization .

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“…Unlike PARP1, PCNA is slowly recruited to DNA damage sites reaching maximum intensity 4–6 min after microirradiation [33,34]. PCNA remains stably bound at DNA damage sites to coordinate timely recruitment of DNA replication and repair factors [6,33,34].…”

“…PCNA remains stably bound at DNA damage sites to coordinate timely recruitment of DNA replication and repair factors [6,33,34]. As a ring-shaped homotrimer, PCNA encircles and slides along DNA, and recruits proteins via the interdomain connector loop on the outer surface serving as the common PCNA-protein interaction interface [14].…”

Creating localized DNA double-strand breaks with microirradiation

“…A number of techniques have been developed to fulfill this purpose, however, all these methods require laser sources or other specific devices. For example, a study using an ultraviolet A (UVA) laser in combination with BrdU-labelling stimulated experiments using UVA lasers of different wavelengths to examine the spaciotemporal dynamics of the factors involved in DNA damage response and DNA damage repair (8)(9)(10)(11)(12)(13)(14). More recently, green and near-infrared lasers, which do not require BrdU-labelling, have been applied (15)(16)(17).…”