RNF8 explained

E3 ubiquitin-protein ligase RNF8 is an enzyme that in humans is encoded by the RNF8 gene.^{[1] [2] [3]} RNF8 has activity both in immune system functions^[4] and in DNA repair.

Function

The protein encoded by this gene contains a RING finger motif and an FHA domain. This protein has been shown to interact with several class II ubiquitin-conjugating enzymes (E2), including UBE2E1/UBCH6, UBE2E2, and UBE2E3, and may act as a ubiquitin ligase (E3) in the ubiquitination of certain nuclear proteins. Alternatively spliced transcript variants encoding distinct isoforms have been reported.^[3]

RNF8 promotes repair of DNA damage through three DNA repair pathways: homologous recombinational repair (HRR),^[5] non-homologous end joining (NHEJ),^{[6] [7]} and nucleotide excision repair (NER).^[6] DNA damage is considered to be the primary cause of cancer, and deficiency in DNA repair can cause mutations leading to cancer.^[8] A deficiency in RNF8 predisposes mice to cancer.^{[9] [10]}

Chromatin remodeling

After the occurrence of a double-strand break in DNA, the chromatin needs to be relaxed to allow DNA repair, either by HRR or by NHEJ. There are two pathways that result in chromatin relaxation, one initiated by PARP1 and one initiated by γH2AX (the phosphorylated form of the H2AX protein) (see Chromatin remodeling). Chromatin remodeling initiated by γH2AX depends on RNF8, as described below.

The histone variant H2AX constitutes about 10% of the H2A histones in human chromatin.^[11] At the site of a DNA double-strand break, the extent of chromatin with phosphorylated γH2AX is about two million base pairs.

γH2AX does not, by itself, cause chromatin decondensation, but within seconds of irradiation the protein “Mediator of the DNA damage checkpoint 1” (MDC1) specifically attaches to γH2AX.^{[12] [13]} This is accompanied by simultaneous accumulation of RNF8 protein and the DNA repair protein NBS1 which bind to MDC1.^[14] RNF8 mediates extensive chromatin decondensation through its subsequent interaction with CHD4 protein,^[15] a component of the nucleosome remodeling and deacetylase complex NuRD.

RNF8 in Homologous Recombinational Repair

DNA end resection is a pivotal step in HRR repair that produces 3’ overhangs that provide a platform to recruit proteins involved in HRR repair. The MRN complex, consisting of Mre11, Rad50 and NBS1, carries out the initial steps of this end resection.^[16] RNF8 ubiquitinates NBS1 (both before and after DNA damage occurs), and this ubiquitination is required for effective homologous recombinational repair. Ubiquitination of NBS1 by RNF8 is, however, not required for the role of NBS1 in another DNA repair process, the error-prone microhomology-mediated end joining DNA repair.

RNF8 appears to have other roles in HRR as well. RNF8, acting as a ubiquitin ligase, mono-ubiquitinates γH2AX to tether DNA repair molecules at DNA lesions.^[17] In particular, RNF8 activity is required to recruit BRCA1 for homologous recombination repair.^[18]

RNF8 in Non-Homologous End Joining

Ku protein is a dimeric protein complex, a heterodimer of two polypeptides, Ku70 and Ku80. Ku protein forms a ring structure. An early step in non-homologous end joining DNA repair of a double-strand break is the slipping of a Ku protein (with its ring protein structure) over each end of the broken DNA. The two Ku proteins, one on each broken end, bind to each other and form a bridge.^{[19] [20]} This protects the DNA ends and forms a platform for further DNA repair enzymes to operate. After the broken ends are rejoined, the two Ku proteins still encircle the now intact DNA and can no longer slip off an end. The Ku proteins must be removed or they cause loss of cell viability.^[21] The removal of Ku protein is performed either by RNF8 ubiquitination of Ku80, allowing it to be released from the Ku protein ring,^[22] or else by NEDD8 promoted ubiquitination of Ku protein, causing its release from DNA.

RNF8 in Nucleotide Excision Repair

UV-induced formation of pyrimidine dimers in DNA can lead to cell death unless the lesions are repaired. Most repair of these lesions is by nucleotide excision repair.^[23] After UV-irradiation, RNF8 is recruited to sites of UV-induced DNA damage and ubiquitinates chromatin component histone H2A. These responses provide partial protection against UV irradiation.^{[6] [24]}

Impaired spermatogenesis

Spermatogenesis is the process in which spermatozoa are produced from spermatogonial stem cells by way of mitosis and meiosis. A major function of meiosis is homologous recombinational repair of this germline DNA. RNF8 plays an essential role in signaling the presence of DNA double-strand breaks. Male mice with a gene knockout for RNF8 have impaired spermatogenesis, apparently due to a defect in homologous recombinational repair.

Interactions

RNF8 has been shown to interact with Retinoid X receptor alpha.^[25]

See also

• RING finger domain

Further reading

• Maruyama K, Sugano S . Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides . Gene . 138 . 1–2 . 171–4 . Jan 1994 . 8125298 . 10.1016/0378-1119(94)90802-8 .
• Bonaldo MF, Lennon G, Soares MB . Normalization and subtraction: two approaches to facilitate gene discovery . Genome Research . 6 . 9 . 791–806 . Sep 1996 . 8889548 . 10.1101/gr.6.9.791 . free .
• Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A, Sugano S . Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library . Gene . 200 . 1–2 . 149–56 . Oct 1997 . 9373149 . 10.1016/S0378-1119(97)00411-3 .
• Ito K, Adachi S, Iwakami R, Yasuda H, Muto Y, Seki N, Okano Y . N-Terminally extended human ubiquitin-conjugating enzymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8 . European Journal of Biochemistry . 268 . 9 . 2725–32 . May 2001 . 11322894 . 10.1046/j.1432-1327.2001.02169.x . free .
• Takano Y, Adachi S, Okuno M, Muto Y, Yoshioka T, Matsushima-Nishiwaki R, Tsurumi H, Ito K, Friedman SL, Moriwaki H, Kojima S, Okano Y . The RING finger protein, RNF8, interacts with retinoid X receptor alpha and enhances its transcription-stimulating activity . The Journal of Biological Chemistry . 279 . 18 . 18926–34 . Apr 2004 . 14981089 . 10.1074/jbc.M309148200 . free .
• Lehner B, Sanderson CM . A protein interaction framework for human mRNA degradation . Genome Research . 14 . 7 . 1315–23 . Jul 2004 . 15231747 . 442147 . 10.1101/gr.2122004 .
• Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP, Vidal M . Towards a proteome-scale map of the human protein-protein interaction network . Nature . 437 . 7062 . 1173–8 . Oct 2005 . 16189514 . 10.1038/nature04209 . 2005Natur.437.1173R . 4427026 .
• Kolas NK, Chapman JR, Nakada S, Ylanko J, Chahwan R, Sweeney FD, Panier S, Mendez M, Wildenhain J, Thomson TM, Pelletier L, Jackson SP, Durocher D . Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase . Science . 318 . 5856 . 1637–40 . Dec 2007 . 18006705 . 2430610 . 10.1126/science.1150034 . 2007Sci...318.1637K .

Notes and References

1. Ishikawa K, Nagase T, Suyama M, Miyajima N, Tanaka A, Kotani H, Nomura N, Ohara O . Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro . DNA Research . 5 . 3 . 169–76 . Jun 1998 . 9734811 . 10.1093/dnares/5.3.169 . free .
2. Seki N, Hattori A, Sugano S, Suzuki Y, Nakagawara A, Ohhira M, Muramatsu M, Hori T, Saito T . Isolation, tissue expression, and chromosomal assignment of a novel human gene which encodes a protein with RING finger motif . Journal of Human Genetics . 43 . 4 . 272–4 . Jan 1999 . 9852682 . 10.1007/s100380050088 . free .
3. Web site: Entrez Gene: RNF8 ring finger protein 8.
4. Ramachandran S, Chahwan R, Nepal RM, Frieder D, Panier S, Roa S, Zaheen A, Durocher D, Scharff MD, Martin A . The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination . Proc. Natl. Acad. Sci. U.S.A. . 107 . 2 . 809–14 . 2010 . 20080757 . 2818930 . 10.1073/pnas.0913790107 . 2010PNAS..107..809R . free .
5. Lu CS, Truong LN, Aslanian A, Shi LZ, Li Y, Hwang PY, Koh KH, Hunter T, Yates JR, Berns MW, Wu X . The RING finger protein RNF8 ubiquitinates Nbs1 to promote DNA double-strand break repair by homologous recombination . J. Biol. Chem. . 287 . 52 . 43984–94 . 2012 . 23115235 . 3527981 . 10.1074/jbc.M112.421545 . free .
6. Marteijn JA, Bekker-Jensen S, Mailand N, Lans H, Schwertman P, Gourdin AM, Dantuma NP, Lukas J, Vermeulen W . Nucleotide excision repair-induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response . J. Cell Biol. . 186 . 6 . 835–47 . 2009 . 19797077 . 2753161 . 10.1083/jcb.200902150 .
7. Feng L, Chen J . The E3 ligase RNF8 regulates KU80 removal and NHEJ repair . Nat. Struct. Mol. Biol. . 19 . 2 . 201–6 . 2012 . 22266820 . 3888515 . 10.1038/nsmb.2211 .
8. Kastan MB . DNA damage responses: mechanisms and roles in human disease: 2007 G.H.A. Clowes Memorial Award Lecture . Molecular Cancer Research . 6 . 4 . 517–24 . April 2008 . 18403632 . 10.1158/1541-7786.MCR-08-0020 . free .
9. Li L, Halaby MJ, Hakem A, Cardoso R, El Ghamrasni S, Harding S, Chan N, Bristow R, Sanchez O, Durocher D, Hakem R . Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer . J. Exp. Med. . 207 . 5 . 983–97 . 2010 . 20385750 . 2867283 . 10.1084/jem.20092437 .
10. Halaby MJ, Hakem A, Li L, El Ghamrasni S, Venkatesan S, Hande PM, Sanchez O, Hakem R . Synergistic interaction of Rnf8 and p53 in the protection against genomic instability and tumorigenesis . PLOS Genet. . 9 . 1 . e1003259 . 2013 . 23382699 . 3561120 . 10.1371/journal.pgen.1003259 . free .
11. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM . DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139 . J. Biol. Chem. . 273 . 10 . 5858–68 . 1998 . 9488723 . 10.1074/jbc.273.10.5858. free .
12. Mailand N, Bekker-Jensen S, Faustrup H, Melander F, Bartek J, Lukas C, Lukas J . RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins . Cell . 131 . 5 . 887–900 . 2007 . 18001824 . 10.1016/j.cell.2007.09.040 . free .
13. Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP . MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks . Cell . 123 . 7 . 1213–26 . 2005 . 16377563 . 10.1016/j.cell.2005.09.038 . free .
14. Chapman JR, Jackson SP . Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage . EMBO Rep. . 9 . 8 . 795–801 . 2008 . 18583988 . 2442910 . 10.1038/embor.2008.103 .
15. Luijsterburg MS, Acs K, Ackermann L, Wiegant WW, Bekker-Jensen S, Larsen DH, Khanna KK, van Attikum H, Mailand N, Dantuma NP . A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure . EMBO J. . 31 . 11 . 2511–27 . 2012 . 22531782 . 3365417 . 10.1038/emboj.2012.104 .
16. Liu T, Huang J . DNA End Resection: Facts and Mechanisms . Genomics Proteomics Bioinformatics . 14 . 3 . 126–30 . 2016 . 27240470 . 4936662 . 10.1016/j.gpb.2016.05.002 .
17. Yamamoto T, Taira Nihira N, Yogosawa S, Aoki K, Takeda H, Sawasaki T, Yoshida K . Interaction between RNF8 and DYRK2 is required for the recruitment of DNA repair molecules to DNA double-strand breaks . FEBS Lett. . 591 . 6 . 842–853 . 2017 . 28194753 . 10.1002/1873-3468.12596 . free .
18. Hodge CD, Ismail IH, Edwards RA, Hura GL, Xiao AT, Tainer JA, Hendzel MJ, Glover JN . RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment . J. Biol. Chem. . 291 . 18 . 9396–410 . 2016 . 26903517 . 10.1074/jbc.M116.715698 . 4850281. free .
19. Jones JM, Gellert M, Yang W . A Ku bridge over broken DNA . Structure . 9 . 10 . 881–4 . 2001 . 11591342 . 10.1016/s0969-2126(01)00658-x. free .
20. Rulten SL, Grundy GJ . Non-homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process . BioEssays . 39 . 3 . 1600209. 2017 . 28133776 . 10.1002/bies.201600209 . 205477344 .
21. Brown JS, Lukashchuk N, Sczaniecka-Clift M, Britton S, le Sage C, Calsou P, Beli P, Galanty Y, Jackson SP . Neddylation promotes ubiquitylation and release of Ku from DNA-damage sites . Cell Rep . 11 . 5 . 704–14 . 2015 . 25921528 . 4431666 . 10.1016/j.celrep.2015.03.058 .
22. Postow L, Ghenoiu C, Woo EM, Krutchinsky AN, Chait BT, Funabiki H . Ku80 removal from DNA through double strand break-induced ubiquitylation . J. Cell Biol. . 182 . 3 . 467–79 . 2008 . 18678709 . 2500133 . 10.1083/jcb.200802146 .
23. Douki T, von Koschembahr A, Cadet J . Insight in DNA Repair of UV-induced Pyrimidine Dimers by Chromatographic Methods . Photochem. Photobiol. . 93 . 1 . 207–215 . 2017 . 27935042 . 10.1111/php.12685 . free .
24. Sakasai R, Tibbetts R . RNF8-dependent and RNF8-independent regulation of 53BP1 in response to DNA damage . J. Biol. Chem. . 283 . 20 . 13549–55 . 2008 . 18337245 . 10.1074/jbc.M710197200 . free .
25. Takano Y, Adachi S, Okuno M, Muto Y, Yoshioka T, Matsushima-Nishiwaki R, Tsurumi H, Ito K, Friedman SL, Moriwaki H, Kojima S, Okano Y . The RING finger protein, RNF8, interacts with retinoid X receptor alpha and enhances its transcription-stimulating activity . The Journal of Biological Chemistry . 279 . 18 . 18926–34 . Apr 2004 . 14981089 . 10.1074/jbc.M309148200 . free .