The Pso4 mRNA Splicing and DNA Repair Complex Interacts with WRN for Processing of DNA Interstrand Cross-links

Abstract

DNA interstrand cross-links (ICLs) are perhaps the most formidable lesion encountered by the cellular DNA repair machinery, and the elucidation of the process by which they are removed in eukaryotic cells has proved a daunting task. In particular, the early stages of adduct recognition and uncoupling of the cross-link have remained elusive principally because genetic studies have not been highly revealing. We have developed a biochemical assay in which processing of a DNA substrate containing a site-specific psoralen ICL can be monitored in vitro. Using this assay we have shown previously that the mismatch repair factor MutS␤, the nucleotide excision repair heterodimer Ercc1-Xpf, and the replication proteins RPA and PCNA are involved in an early stage of psoralen ICL processing. Here, we report the identification of two additional factors required in the ICL repair process, a previously characterized pre-mRNA splicing complex composed of Pso4/Prp19, Cdc5L, Plrg1, and Spf27 (Pso4 complex), and WRN the protein deficient in Werner syndrome. Analysis of the WRN protein indicates that its DNA helicase function, but not its exonuclease activity, is required for ICL processing in vitro. In addition, we show that WRN and the Pso4 complex interact through a direct physical association between WRN and Cdc5L. A putative model for uncoupling of ICLs in mammalian cells is presented.

Double-strand breaks induce homologous recombinational repair of interstrand cross-links via cooperation of MSH2, ERCC1-XPF, REV3, and the Fanconi anemia pathway

“…Our findings, reported here demonstrate that the mismatch repair factor MSH2 acts, presumably in concert with ERCC1-XPF, to uncouple the ICL [21]. Our previous findings have also implicated RPA, WRN, and a complex composed of PRP19, CDC5L, PLRG1, and SPF27 in the uncoupling process [34,37]. Subsequent to the uncoupling of the ICL, REV3 would act to bypass the remaining monoadduct.…”

“…In addition, MSH2 and ERCC1-XPF have been shown to interact by co-IP experiments [26]. We have also shown an involvement of RPA, WRN, and a four protein complex composed of PRP19/PSO4, CDC5L, SPF27, and PLRG1 in ICL repair processing using the CRS assay mentioned above [34]. The CRS assay measures the initial recognition and processing of the ICL, and thus the factors required for this assay are involved in the uncoupling stage of repair.…”

The Prp19/Pso4 core complex undergoes ubiquitylation and structural alterations in response to DNA damage

“…siRNAs were purchased from Dharmacon. The sequence of the hPrp19 siRNA was previously described [21]. For treatment with DNA damaging agents, methyl-methane sulfonate (MMS) or mitomycin C (MMC) was added to the medium 24 hours after transfection.…”

“…HeLa whole cell extracts were prepared and fractionated as described [21,22]. For responses to DNA damage, HeLa cells were transfected with the indicated plasmid, and MMS was added to the medium 24 hours later.…”

“…Furthermore, the human protein has also been shown to interact with terminal deoxynucleotidyl transferase, and to be involved in mediating cell survival after DNA damage [20]. In addition, we have recently demonstrated a biochemical role for the core complex in processing of ICLs in vitro, and showed a direct physical interaction between Cdc5L and WRN [21]. These findings indicate that the Prp19/Pso4 core complex has a direct role in mediating the cellular response to DNA damage in addition to its demonstrated role in pre-mRNA splicing.…”