The kinase ATR (ATM and Rad3‑ related) and its regulators, including ATRIP (ATR‑interacting protein), are recruited to RPA‑coated ssDNA (replication protein A–single‑stranded DNA; an intermediate in DNA repair and the response to replication stress) to coordinate the DNA damage response (DDR). Maréchal et al. now show that following DNA damage, the E3 ubiquitin ligase pre‑mRNA‑processing facto r 19 (PRP19) ubiquitylates RPA to promote the accumulation of ATR–ATRIP complexes at RPA–ssDNA and thus the DDR.

The authors carried out a bio‑chemical screen for proteins that bind to RPA–ssDNA, identifying 340 candidates. Of these they focused on PRP19, which contains a U‑box and forms a complex with CDC5L, PRL1 and SPF27 — this complex is known to have a role in pre‑mRNA splicin g. All four complex components bound wild‑type RPA–ssDNA, but not ssDNA bound by RPA that is defective for the DDR. This, coupled with the fact that PRP19 has previously been shown to be important for genomic stability, prompted the authors to ask whether PRP19 has a role in the DDR.

They found that PRP19 bound RPA in cells following DNA dam‑age. This interaction was dependent on Tyr405 in the WD40 domain of PRP19 and enhanced by ATR activ‑ity. Importantly, although PRP19 was localized to nuclear speckles in unstressed cells, consistent with its role in splicing, it colocalized with RPA32 (as did CDC5L) in γH2A.X stripes following microirradiation‑induced DNA damage — a range of DDR proteins are known to localize to such stripes. Moreover, a PRP19 mutant that cannot bind RPA failed to localize to DNA damage. Thus, PRP19 senses DNA damage through its interaction with RPA.

So, what is the functional role of PRP19 in the DDR? Knockdown of PRP19 or CDC5L reduced the level of ATR‑induced RPA32 and checkpoint kinase 1 (CHK1) phos‑phorylation after DNA damage, inde‑pendently of the splicing function of PRP19. Furthermore, cells depleted of PRP19 or CDC5L and treated with

DNA‑damaging agents took longer to recover from replication arrest, and harboured slower replication forks and more prematurely terminated replication tracts (which are indica‑tive of stalled or collapsed forks) than controls. Thus, the PRP19 complex seems to regulate ATR activity in response to DNA damage to promote DNA replication.

Finally, to confirm which activity of PRP19 is required for its role in the DDR, the authors depleted cells of the endogenous protein and replaced it with wild‑type PRP19, PRP19 that cannot bind RPA or a ligase‑inactive PRP19. Only wild‑type PRP19 restored RPA32 and CHK1 phos‑phorylation following DNA damage, which suggests that PRP19 senses RPA–ssDNA by binding to RPA and functions as an E3 ligase to promote ATR activation. Additional experi‑ments revealed that PRP19 promotes the attachment of Lys63‑linked ubiq‑uitin chains to RPA32 in response to DNA damage, which promotes ubiquitin chain–ATRIP interactions and the subsequent recruitment of ATR–ATRIP complexes to sites of DNA damage where ATR is then activated.

In short, this study delineates the previously elusive role of PRP19 in the DDR, confirming how it regulates genome stability in addition to its role in RNA processing.

Katharine H. Wrighton

ORIGINAL RESEARCH PAPER Maréchal, A. et al. PRP19 transforms into a sensor of RPA–ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry. Mol. Cell http://dx.doi.org/10.1016/j.molcel.2013.11.002 (2013)

PRP19 promotes the attachment of Lys63‑linked ubiquitin chains to RPA32 in response to DNA damage

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A ligase makes sense of DNA damage

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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 14 | FEBRUARY 2014

Nature Reviews Molecular Cell Biology | AOP, published online 17 January 2014; doi:10.1038/nrm3739

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