Meeting Report. Gordon Research Conferences Mammalian DNA Repair. From 8/Feb/2009 to 13/Feb/2009 at Ventura,CA,USA

Transcription

1 Meeting Report Affiliation Radiation Genetics Name Junko IIJIMA Name of the meeting Period venue and Type of your presentation Title of your presentation Gordon Research Conferences Mammalian DNA Repair From 8/Feb/2009 to 13/Feb/2009 at Ventura,CA,USA 1) Invited lecture 2) Poster presentation 3) Workshop / Symposium 4) Others (Please explain specifically below ( ) RAP80 works to repair of topoisomerase II-mediated DNA damage 1. Summary of your presentation (Include what you learned from discussions with audience) It was reported that RAP80 is included in two distinct BRCA1 complexes, A-complex (BRCA1-Abraxas-Rap80) and C-complex (BRCA1-CtIP-Rap80), and is required for the recruit of BRCA1 to irradiation-induced DNA damage sites (Science 316,2007). In this study, we disrupted the RAP80 gene in chicken DT40 cells to evaluate roles of RAP80 in DNA damage repair. RAP80-/- cells showed sensitivity to topoisomerase II poison like VP16 and XK469 that stabilize topoisomerase II-DNA cleavage complex. Interestingly, RAP80-/- cells did not indicate sensitivity to ICRF 193, Topoisomerase II catalytic inhibitor, that dose not make topoisomerase II-DNA cleavage complex. Furthermore, we found that RAP80 is not necessary for homologous recombination (HR) or non homologous end joining (NHEJ), two major pathways for DNA double strand break (DSB) repair. Therefore, we conclude that chicken RAP80 plays a role in removing stabilized Topoisomerase II from ends of DNA. It was reported that Rap80 is included in CtIP-BRCA1 complex and we showed that pg. 1

2 RAP80-/- cells have similar character to CtIP-/- cells. These results suggest that Rap80 works to repair topoisomerase II-induced DNA damage in CtIP-dependent pathway. pg. 2

3 2. Other topics of your interest 1) I was interested in the Junjie Chen s (Yale University School of Medicine, USA) report about MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kd). Brest cancer susceptibility gene 1, BRCA1, plays role in the maintenance of genomic integrity. BRCA1 forms multiprotein complex (BRCA1-BIRD1-Abraxas-Rap80-BRCC36). He described a novel member of the complex, MERIT40. Rap80, the member of the complex, recognizes the ubiquitinated proteins at DNA damage sites and recruits BRCA1 at the sites. MERIT40 also localizes to DNA damage sites and facilitates BRCA1 localization via a role in maintaining the stability of the complex. 2) Abdallah Alhakim (Samuel Lunenfeld Research Institute, Canada) indicated that the RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties) syndrome Protein Mediates a Ubiquitin-Dependent Signaling Cascade at sites of DNA damage. They identified the gene mutated in RIDDLE syndrome, RNF168. RNF168 is an E3 ubiquitin ligase. RNF168 acts with E2 ubiqitin conjugating enzyme UBC 13 to promote the ubiquitination of H2AX histones at DNA damage sites. RNF168 facilitated accumulation of BRCA1 and RAP80 at DNA lesions. They suggest that RNA168-dependent ubiquitylation mediates the accumulation of BRCA1 multiple complex (I described the complex in report 2-1) at DNA damage sites to promote DNA repair. 3) pg. 3

4 Tanya Paull (University of Texas at Austin, USA) reported that Mre11/Rad50 complex acts cooperatively with helicase HerA and a 5 to 3 exonuclease NurA to resect the 5 strand at a DNA end. The Mre11/Rad50 complex works in the early steps of DNA double strand break (DSB) repair. The enzymatic character of this complex, 3 to 5 exonuclease, is polarity paradox because initial step of DNA DSB repair shows DNA 5 strand resection. They proved that Mre11/Rad50 acts cooperatively with HerA and NurA to resect DNA 5 strand end by Nuclease Assay with labeled DNA substrates and purified proteins. They proposes the model that the Mre11/Rad50 complex binds to DNA DSB ends and processes DNA 3 ends and the HerA/NurA complex resects 5 strand to induce DNA repair. 4) Satoshi Tateishi (IMEG Kumamoto University, JAPAN) showed that Rad 18 is required for long-term maintenance of spermatogenesis in mouse testes. DNA lesions during DNA replication cause stalling of the replication fork and results in cell death. DNA postreplication repair (PRR) the DNA repair mechanism that resumes DNA synthesis without removing the DNA replication blocking lesions and repair the DNA lesions after replication. Rad18 is an E3 ubiquitin ligase and plays roles in PRR. Previously, it is reported that the Rad18 was highly expressed in mouse pachytene spermatocytes of the testes in adult mice. S.Tateishi et al generated Rad18-/- mice to clear the Rad18 function in testes. Interestingly, however Rad18-/- male mice were fertile, fertility decreased with age because of the depletion of germ line cells in seminiferous tubules. Therefore, they concluded that Rad18 is required for the long-term maintenance of spermatogenesis in mouse testes. pg. 4

5 5) Stephen Jackson (Cancer Research UK Gurdon Institute, UK) reported about analysis of human CtIP Thr-847 mutants. His group had studied the mechanism of DNA-end resection during cell cycle and reported the resection is controlled by CtIP via phosphorylation of CtIP Thr-847 in cyclin dependent kinase (CDK) dependent manner. CtIP controls DNA double strand break resection. In their study, they mutated CtIP Thr-847, a highly concerved cyclin dependent kinase (CDK) target site, and showed Thr847Ala impairs DNA resection. However, CtIP Thr847Glu mutant promoted resection even after CDK inhibition. These results suggest CDK-mediated phosphorylation of CtIP Thr-847 controls DSB resection and effects DNA DSB repair. pg. 5

6 3. Impression of the meeting you got (e.g., major trends in the field, status and contribution of your study and/or studies in Japan to the field, etc.) The Gordon Research Conference mammalian DNA repair was my first one that I attended to present my data. This conference focused on the mechanisms of DNA repair, especially on the crosstalk among multiple repair pathways, regulation by post-translational modifications, and the development of new biomarkers and therapeutic agents in cancer therapy. Many lab heads and specialist participated in the conference, and we had enough time to discuss our experiment with them. In this meeting, I presented my unpublished observations on a role of the ubiquitin-binding protein RAP80 in DNA repair. It is reported that Rap80 was included in two distinct complexes,brca1-abraxas complex and BRCA1-CtIP complex. Junjie Chen (Yale University School of Medicine, USA) showed MERIT40 as a new member of BRCA1-Abraxas-Rap80 complex. The role of this complex is being clarified however it keeps unclear how the BRCA1-CtIP-Rap80 complex works to repair DNA damage. We found that chicken RAP80-/- cells showed sensitivity to topoisomerase II poisons but not to other agents. This character of RAP80-/- DT40 cells similar to CtIP-/- DT40 cells. RAP80 -/- cells showed normal homologous recombination and non homologous end joining. Chicken RAP80 plays a role in removing stabilized Topoisomerase II from ends of DNA with CtIP. Tanya Paull (University of Texas at Austin, USA) indicated the mechanism that Mre11/Rad 50 resects DNA 5 strand end by biochemical assay. Stephen Jackson (Cancer Research UK Gurdon Institute, UK) studied CtIP-dependent DNA resection by observation of RPA focus formation which happens following DNA resection. Their experiments gave me hints how to prove our hypothesis that Rap80-CtIP may play a role to remove stabilized topoisomerase II form end of DNA. pg. 6

7 pg. 7