Font Size: a A A

Mechanism Of Homologous Recombination Mediated By Argonaute2and DNA Double-Strand Break-Induced Small Rnas

Posted on:2014-01-04Degree:DoctorType:Dissertation
Country:ChinaCandidate:W WeiFull Text:PDF
GTID:1220330401455790Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
DNA double-strand breaks (DSBs) are deleterious DNA damage forms that can lead to gene mutations, genome instability and even cell death, if cannot be properly repaired. Eukaryotes have thus evolved complex mechanisms to efficiently repair DSBs. DSB triggers a complex network of processes in cells termed DNA damage response (DDR), which requires coordinated enzymatic actions of protein sensor, transducer, and effectors in DSB signaling cascade to detect and repair DSB. Small RNAs (sRNAs) are19-30nucleotide (nt) non-coding RNAs. They have emerged to play fundamental roles in various biological processes through regulating gene expression at the transcriptional or post-transcriptional level. One of the common themes in small RNA action is that small RNA should associate with specific Argonaute (Ago) proteins to form the RNA-induced silencing complex (RISC) to exert its function.Recently we and others have discovered a novel class of small RNAs (DSB-induced small RNAs, diRNAs) that are produced from sequences around DSB sites. diRNAs are conserved in plant, mammal and fly, and important for efficient DSB repair. However, the mechanism through which they function in DSB repair remains unclear. Here we utilize an integrated approach involving biochemistry and cell biology technologies to dissect the mechanism through which diRNA act in human cells. We show that diRNAs functioning in DSB repair is restricted to repair by homologous recombination (HR) but not by non-homologous end joining (NHEJ). The role of diRNAs in DSB repair specifically relays on the effector protein Ago2as well as RNase III protein Dicer in mammalian cells. We further demonstrate that Ago2forms a complex with Rad51, a key recombinase in HR repair. The interaction is enhanced in cells treated with ionizing radiation and depends on activities of two PI3kinases, ATM and ATR. Ago2is required for recruiting Rad51to DSB sites but does not affect the accumulation of proteins upstream of Rad51as well as the formation of ssDNA. Although diRNAs are dispensable for the interaction between Ago2and Rad51, diRNA-bound Ago2is indispensable for efficient accumulation of Rad51at DSB sites and hence DSB repair through HR. Our findings suggest that in association with diRNAs generated around from DSB sites, Ago2recruits Rad51to the DSB sites to facilitate repair by HR Taken together, we revealed the mechanisms that how diRNAs, as novel sRNA molecules, function in DSB repair. Our findings not only expand the roles of sRNAs, but also at the first time add a new layer of RNA components to the DSB repair pathway. Our findings greatly expand the knowledge of both sRNA field and DNA damage repair field, and provide new insights into studying the novel functions of sRNA and the underlying mechanisms of DSB repair.
Keywords/Search Tags:DSB repair, small RNAs, homologous recombination, diRNA, Argonaute2
PDF Full Text Request
Related items