Note for RNF138
Two papers that I have read; one is the short communication which briefs the major finding of RNF138 contributing to DNA repair. The other paper is the original paper which performed the experiments to show that RNF138 is the new finding regulator of HR.
Note for –
2. The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice (doi: 10.1038/ncb3259)
RNF138 will be a good target for the choice switching between HR and NHEJ.
Protein modification by ubiquitin has a central role in regulating DSB repair.
Each cell cycle fate, will be controlled by different sets of protein through the ubiquitination process, like in this case, it is showed that RNF138 promote Ku ubiquitination during S and G2 phase when resection is operational.
Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. This study identifies RNF138 (Ub-E3 ligase) which regulates HR pathway. RNF138 has the zinc finger protein which later is found that it can bind with 5' or 3' single-stranded overhangs.
RNF138;
1. stimulate end resection
2. promote ATR-dependent signalling
3. promote DSB by HR
4. propose RNF138 can remove Ku (besides Parp1 which has been reported to regulate Ku and initiate the HR)
I think this is the first report show the new protein, RNF138 can regulate DSB repair pw choice.
NHEJ is active throughout the cell cycle, but is most important during G1 before chromatid replication.
DNA end resection is a key step at which repair pathway choice', the choice between NHEJ and HR, is determined because the generation of ssDNA would favour HR and concomitantly prevent the binding of NHEJ proteins (my guess would be because of the protein recognize the end resection differently).
However, if the MRN complex alone was sufficient to displace Ku from DNA ends, the high abundance and DSB binding of the MRN complex throughout the cell cycle could be problematic in G1, where NHEJ is preferred (or else it would find the homology sequence from another chromosome to pair with).
we demonstrate that Nemo-like kinase (NLK)-associated ring finger protein (NARF) or RNF138 is a ubiquitin E3 ligase that regulates DNA end resection and HR and that this regulation involves direct interaction with and ubiquitylation of Ku80.
Underline assumption -- Ubiquitinylation regulates the HR repair as well as pathway of choice -->, therefore, the group using the siRNA screening library targeting Ubiquitinylation process and using the Rad52 as the readout platform -- to relate it involves in DSB repair - the team used CPT to generate the DSB at S-phase.
How do they identify the regulator of HR repair?
1. using the short interfering RNA (siRNA) library targeting putative members of E3 ubiquitin ligase in U2OS cells which contain Rad52-GFP and H2B-Red fluorescence
2. using Rad52 foci formation as HR readout -- to identify new E3 ligase.
A Rad52-based reporter system was previously used to quantify the relative contribution of HR at different points of the cell cycle in single cells. Rad52 is a recombinase mediator protein that forms foci at DSB sites that are committed to homology-dependent repair. RFP-H2B fluorescence was used to identify the cell nucleus.
Expected outcome;
· depletion in E3 ubiquitin ligase involved in HR repair would inhibit camptothecin (CPT)-induced GFP-Rad52 foci.
Kind of wonder, if we perform exactly the same experiment but different cell lines, will it get the different set of E3 ubiquitin ligase that regulate the HR?
Known E3 ubiquitin ligase that regulate DSB repair;
1.RNF4
2.CtIF
3.BRCA1
1. obtain the result from library screening -- get the set of some genes (using CPT - to specifically looking for the one that affect HR)
2. confirm the result by looking at Rad51 foci formation (using IF)
3. experiment show RNF138 is recruited to DSB site
4. elucidate more and show that ZF in RNF138 is responsible for recruitment
RNF138 can be found in most eukaryotes except yeast.
From genome-wide screen;
RNF138 was phosphorylated following DNA damage by ATM/ATR
Although we could not detect RNF138 recruitment to ionizing-radiation-induced foci, we found that GFP RNF138 is readily recruited to and retained at DNA damage sites after laser irradiation.
We took advantage of a previously established DSB reporter system that uses a LacI-FokI nuclease fusion protein to create DSBs within a single genomic locus in U2OS cells (U2OS DSB reporter).
There are three experiment to confirm the recruitment to DSB (1 fail and the other two successes -- it requires enrichment to see the phenotype)
1. IF after ionizing-radiation (not success)
2. laser irradiation -- using RNF138-GFP --> be able to observe the foci
3. using DSB reporter system -- LacI-FokI nuclease to generate DSB (like TALENT and ZF) + chromatin immunoprecipitation
The recruitment of RNF138 to sites of DNA breaks is mediated by its zinc finger motifs.
To explore how RNF138 might regulate the HR repair pathway -- they use HEK293 cell line, RNF138-GFP tag --> immunoprecipitation --> Mass spec --> found out that DNA-PKs, Ku70, Ku80 interacting with RNF138.
After they find the binding partner then they investigate more whether those binding partners are ubiquitinylated by RNF138. Ku80 is the substrate for the RNF138 after the DNA damage, only DSB type lesion that stimulate the ubiquitinylation other lesion does not responsible for this action!
There are so many experiments to confirm whether RNF138 can ubiquitinylate Ku80, siRNA, generate mutant which defect in catalytic residues or binding domain so on so forth.
RNF138 stimulates HR whereas RNF8 is reported to stimulate NHEJ.
Thus, our results suggest that RNF8 and RNF138 are the major ubiquitin E3 ligases responsible for Ku80 ubiquitylation but have non-redundant functions restricted to different parts of the cell cycle.
Prevalent model for DSB repair pathway choice involves direct competition for binding between NHEJ (Ku70/Ku80) and HR (MRN/CtIP) determining the extent of DNA end resection.
The inhibition of HR (as measured by the reduction of Rad51 and Rad52 foci) and the observation that RNF138 interacts specifically with Ku heterodimers led us to test the hypothesis that RNF138 stimulates Ku70/80 removal from the break sites to bias repair towards HR.
The requirement of ssDNA overhangs for RNF138 binding to DNA and the defects in Rad51 recruitment to damaged DNA in RNF138- depleted cells prompted us to examine the effect of RNF138 on DNA end resection.
To gain insight into how RNF138 contributes to DSB end resection, we examined whether its depletion affected the recruitment of various factors known to be involved in DNA end resection.
We found that RNF138 is recruited to sites of DSBs through its ZNF domains.
we demonstrated that RNF138 ZNFs contain a robust DNA-binding activity that binds preferentially to resected dsDNA over ssDNA but does not bind blunt-ended dsDNA or PAR.
RNF138 is downstream of Mre11 activity and RNF138-mediated Ku80 ubiquitination decreased Ku binding to chromatin.
RNF138 does not target Ku for proteasome-
mediated degradation, unlike RNF8. Loss of RNF138 reduces HR and stimulates, rather than inhibits, NHEJ.
UBE2D family ubiquitin E2 ligases were identified as the E2 partner of RNF138 in the regulation of HR and CtIP was identified as an RNF138 target.
On the basis of our results, we propose a model for pathway choice in the S/G2 phase. Following a DSB, Ku70/80 heterodimers and the MRN complex are recruited to sites of DNA damage. Ku70/80 binds to DNA damage sites to protect the ends from nonspecific processing. RNF138 then binds specifically to Mre11-resected DNA through its ZNF domains to remove Ku and enable CtIP/Exo1 nuclease recruitment to sites of DSBs for extensive resection and subsequent repair by HR.
Our model assumes that DNA end protection by Ku occurs throughout the cell cycle and is independent of its role in NHEJ repair. This model postulates that if RNF138 is either inhibited or depleted in cells, Ku is not displaced, DNA end resection will be attenuated and HR will not occur.
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