AI Article Synopsis
- RP-A plays a crucial role in DNA replication, repair, and recombination, forming clusters known as foci during S-phase and in response to DNA double-strand breaks.
- Ku86 protein co-localizes with RP-A in these foci, and both proteins facilitate unscheduled DNA synthesis and enhance ligation efficiency in cases of linear double-stranded DNA.
- The study highlights the use of a Xenopus in vitro system to generate DNA double-strand breaks, allowing researchers to investigate RP-A and Ku86 as markers for DNA damage and its repair in conditions mimicking a normal nuclear environment.
Article Abstract
Replication protein A (RP-A) is involved in DNA replication, repair and recombination. It has been demonstrated that RP-A clusters in foci prior to DNA replication and redistributes over chromatin during S-phase. Here, we show that RP-A foci also form in response to DNA double-strand (ds) breaks produced on Xenopus laevis sperm nuclei by restriction enzymes and then reconstituted with Xenopus egg high-speed extracts. Ku86 co-localizes with RP-A in the same foci. An unscheduled RP-A-dependent DNA synthesis takes place overlapping with RP-A and Ku86 foci. Immunoelectron-microscopy analysis reveals that these foci correspond to spherical bodies up to 300 nm in diameter, which contain RP-A, Ku86 and DNA. In an independent in vitro assay, we incubated linear dsDNA bound to magnetic beads with Xenopus egg extracts. Here, also RP-A and Ku cluster in foci as seen through immunofluorescence. Both proteins appear to enrich themselves in sequences near the ends of the DNA molecules and influence ligation efficiency of ds linear DNA to these ends. Thus, the Xenopus in vitro system allows for the generation of specific DNA ds breaks, RP-A and Ku can be used as markers for these lesions and the repair of this type of DNA damage can be studied under conditions of a normal nuclear environment.
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| http://dx.doi.org/10.1242/jcs.114.18.3345 | DOI Listing |
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