Abstract # 2392 The Identification of Mre11 as the Central Participant in Multiple Subpathways of Repairing Chromosomal Double-StrandBreak

Presenter: Zhuang, Jing

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Materials/Methods:
Using a panel of plasmid-based DSB repair substrates which stably integrated into the genome of cultured human cell, HEK293, we analyzed the quantity and quality of the four main DSB repair subpathways. The substrates pDR-GFP and pSSA-GFP were respectively used to assess the two main subtypes of HR processes, homologous­directed repair (HDR) which conducts precise repair, and single-strand annealing (SSA) which repairs DSB with large deletions. Repair of DSB in pDR-GFP through HDR or in pSSA-GFP via SSA reconstituted wild type GFP gene, which can be detected by FACS analysis of GFP expression. The substrates pHW1 was used to quantify and qualify the two major types of NHEJ processes, Ku-dependent conservative NHEJ (C-NHEJ) which represent precise religation in this study, and Ku-independent deletional NHEJ (D-NHEJ) which is imprecise repair using microhomology. Repair of DSB(s) in pHW1 by either C-NHEJ or D-NHEJ will result in expression of functional gpt protein which render cell resistant to the selection of MHATX. The molecular mechanisms of NHEJ used for the repair DSBs (C-NHEJ vs D-NHEJ) were determined by sequence analysis of the end joining junction. Inhibition of Mre11 was achieved by transient expression of siRNA against Mre11. Control siRNA was studied in parallel. Designated enzymatic chromosomal DSBs were induced by transient expression of endonucleases, I-Sce-I.






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