Distinct Roles of FANCO/RAD51C Protein in DNA Damage Signaling and Repair
Authors: Shreelakshmi Subramanya
Kumar Somyajit
Ganesh Nagaraju
Journal: Journal of Biological Chemistry (2012)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: RAD51C; homologous recombination; Fanconi anemia; DNA repair; tumor suppressor
Affiliations: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.

Abstract:  RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor. 

Retracted: No

PubPeer comments:
 ![file] , 
 Figure 1 C - Legend should explain whether these are from Chinese hamster cells or from HeLa cells. The aberration detection is not convinced - arrow indicates a chromosome break rather than a chromatid break (the image is not in high resolution). As far as I can see, there are no radial chromosome in Figure 1 C left panel. , 
 #1 Dear Colleague, We sincerely thank you for pointing out these mistakes. These mistakes must have happened while assembling the first version of the figures (the Main and supplemental figure for this very experiment were assembled together) and later, unfortunately, did not catch our attention. We have all the original files, and we will address this and correct it very soon. We will get back to you very soon. Apologies for such a mistake and many thanks again. We assure you these errors do not affect the final conclusion drawn in this of the paper. Many thanks, Kumar Somyajit , 
 #1 Dear Colleague, u00a0 Please find the correct representative neutral Comet images for the MMC treated experimental sets (Experiments performed- Sept 2011). Due to the size adjustments, images appeared as stretched, but it was just an error of copying while arranging the images for Main + Supplementary Figures. We apologize for this human error. Many thanks for your interest in our work and pointing out these errors, and we really appreciate your efforts. To briefly explain the rationale behind this experiment- we wanted to co-monitor the kinetics of double-strand break (DSB) repair (by Comet assay) and checkpoint signaling (analysis of CHK1 phosphorylation) in cells deficient in RAD51C, as well as the cells expressing various RAD51C pathological variants. Based on the data from this experiment and also various other readouts (Fig: 1,2,4,5,6,7 and 8), we concluded that: 1. RAD51C is a functional component of the FA pathway of ICL repair. u00a0 2. RAD51C contributes to the FA pathway by regulating the homology-directed repair (HDR) at DSBs post-ICL unhooking. 3. RAD51C is required to tightly couple checkpoint signaling (via CHK2 activation) to a DSB repair (via HDR mechanism). This unique function of RAD51C enforces a safeguard mechanism to complete DSB repair before cells enter into mitosis and segregate the damaged chromosomes. u00a0 4. In addition, by employing multiple end-point based readouts, we showed that patient-derived RAD51C variants fall into two categories- deleterious with a complete loss of function (L138F and G125V), and hypomorphic with a partial loss of function (R258H, G264S, R366Q, D159N, and T287A). u00a0 We want to reiterate that the error that occurred during the assembly of comet images for the supplementary material doesnu2019t change the interpretation and conclusions of this paper. We are also approaching the journal for correction of this error. However, meanwhile, If you want to know more or would like to share some insights for this work, please feel free to contact us (kr.somyajit@cpr.ku.dk or nganesh@biochem.iisc.ernet.in). We will be delighted to share any information, data, or reagents. u00a0 Best regards, Kumar and Ganesh ![file] , 
 #2 Our apologies for not directly writing Chinese hamster cells, but we have clearly indicated that these results are obtained from RAD51C deficient CL-V4B cells (Chinese hamster cells). We really found it tough to get good chromosome spreads from hamster cells, and this is the reason that we started analyzing metaphase spreads from HeLa and MCF-7 cells in our further studies. We also agree that the images we obtained were of low resolution (due to several technical issues), but that did not hamper our identifications of chromatid breaks and radial chromosomes. Below is one example that we identified as chromatid breaks or radial chromosomes in our experiments (performed during Aug 2011 for this paper). Many thanks for your interest, and feel free to write to us (kr.somyajit@cpr.ku.dk or nganesh@biochem.iisc.ernet.in ) if you want to discuss more. u00a0 Best regards, Kumar and Ganesh ![file] , 
 2019 correction. http: www.jbc.org content 294 35 13198.short The comet sub-panels in Fig. S4 were erroneously copied during figure assembly. This error has now been corrected using the original files. These changes do not affect the results or conclusions of this work. The authors sincerely apologize for the error. ,