The Pso4 mRNA splicing and DNA repair complex interacts with WRN for processing of DNA interstrand cross-links
Nianxiang Zhang1, Ramandeep Kaur, Xiaoyan Lu
1Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
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 MutSbeta, 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.
Insights
DNA interstrand cross-links (ICLs) pose a significant challenge to DNA repair. This study identifies the Pso4 complex and WRN protein as crucial for ICL processing, revealing WRN
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Biochemistry
Background:
- DNA interstrand cross-links (ICLs) are severe DNA lesions that impede cellular processes.
- Understanding the early stages of ICL recognition and uncoupling in eukaryotes has been challenging due to limited genetic insights.
- Previous research implicated MutSbeta, Ercc1-Xpf, RPA, and PCNA in early psoralen ICL processing.
Purpose of the Study:
- To identify novel factors involved in the in vitro processing of DNA interstrand cross-links.
- To elucidate the specific roles of the WRN protein and the Pso4 complex in ICL repair.
- To investigate the functional domains of WRN required for ICL processing and its interaction with the Pso4 complex.
Main Methods:
- Development of a biochemical assay to monitor the in vitro processing of a psoralen ICL-containing DNA substrate.
- Analysis of the involvement of purified proteins and complexes in the ICL repair assay.
- Biochemical characterization of WRN protein's helicase and exonuclease activities in ICL processing.
- Co-immunoprecipitation assays to determine physical interactions between WRN and Pso4 complex components.
Main Results:
- Identification of the Pso4 complex (Pso4/Prp19, Cdc5L, Plrg1, Spf27) and WRN protein as essential factors for ICL processing.
- Demonstration that WRN's helicase activity, but not its exonuclease activity, is required for in vitro ICL repair.
- Evidence of a direct physical interaction between WRN and Cdc5L within the Pso4 complex.
Conclusions:
- The Pso4 complex and WRN protein are newly identified key players in the mammalian DNA interstrand cross-link repair pathway.
- WRN's helicase function is critical for processing ICLs, suggesting a role in uncoupling the cross-link.
- A model for ICL uncoupling involving the interaction between WRN and the Pso4 complex is proposed.
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