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Fine-tuning the ubiquitin code at DNA double-strand breaks: deubiquitinating enzymes at work.

Citterio E - Front Genet (2015)

Bottom Line: Signaling processes mediated by ubiquitin (ub) are crucial for the cellular response to DNA double-strand breaks (DSBs), one of the most dangerous types of DNA lesions.How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation.One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs), as supported by the implication of a growing number of DUBs in DNA damage response processes.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam Netherlands.

ABSTRACT
Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin (ub) are crucial for the cellular response to DNA double-strand breaks (DSBs), one of the most dangerous types of DNA lesions. In particular, the DSB response critically relies on active ubiquitination by the RNF8 and RNF168 ub ligases at the chromatin, which is essential for proper DSB signaling and repair. How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation. One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs), as supported by the implication of a growing number of DUBs in DNA damage response processes. Here, we discuss the current knowledge of how ub-mediated signaling at DSBs is controlled by DUBs, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer.

No MeSH data available.


Related in: MedlinePlus

The role of deubiquitinating enzymes in the chromatin-based response to DNA double-strand breaks. (A) Recognition of a DNA double-strand break (DSB) by the MRN (MRE11-RAD50-NBS1) complex initiates DDR signaling, triggering ATM (ataxia-telangectasia mutated) kinase-dependent phosphorylation of H2AX (γH2AX). ATM phosphorylates also MDC1 (mediator of DNA damage checkpoint protein 1), which is recognized by the RING finger 8 (RNF8) E3 ligase. The activity of RNF8 is required for recruitment of a second E3, RNF168. RNF168 mono-ubiquitinates H2A-type histones on Lys13 and Lys15 (H2A(X)K13/K15Ub), and the concerted action of RNF8/RNF168 leads to the formation of K63-linked ubiquitin (Ub) chains on these lysines and to ubiquitination of other substrates (Y and W). RNF168 can bind to its own products, thereby amplifying chromatin ubiquitination around the DSB. OTUB1 opposes RNF168 activity in a non-catalytic manner, by binding to the E2 ubiquitin-conjugating enzymes UBC13 and UbcH5. USP3, USP44, and Dub3 DUB activities impair RNF168 recruitment, suggesting that they can target RNF8 substrate(s). These DUBs may also cleave RNF168-mediated ubiquitinated H2A(X). Excessive RNF168-dependent chromatin ubiquitination is limited by the TRIP12 and UBR5 E3 ligases, which target RNF168 for proteasomal degradation. USP34, instead, counteracts DSB-induced RNF168 ubiquitination. DSBs also trigger the recruitment of the Polycomb group E3 RING1B/BMI1, which mono-ubiquitinates H2A on Lys119 (H2AK119Ub) to locally repress transcription. USP16 and BAP1 target the H2AK119Ub mark, and USP16 activity is required for re-activation of DSB-induced transcriptional silencing. USP3 and USP44 oppose to steady-state mono-ubiquitinated H2A, which is primarily constituted by H2AK119Ub. (B) DSB-induced ubiquitin signals are recognized by downstream DDR factors. 53BP1 (p53 binding protein 1) recognizes the H2AK15Ub mark by its UDR (ubiquitination-dependent recruitment) motif, and dimethylated H4K20 (H4K20me2) with its TUDOR domain. One way BRCA1 (Breast cancer 1) is recruited to DSBs is through its interaction with RAP80 (receptor-associated protein 80), a protein that effectively binds K63-ub conjugates. BRCA1 has E3 ligase activity and functions as a heterodimer with BARD1 (BRCA1-associated RING domain protein 1). The DUBs BRCC36, POH1, and OTUB2 regulate DDR signaling by hydrolyzing DSB-induced K63-ub chains. OTUB2 also opposes to RNF8-dependent ubiquitination of L3MBTL1 (not shown). Whether MYSM1, which also possesses K63-ub cleavage activity, participates in this step of DDR is an open question. RNF168-mediated K27-linked ub chains on H2A/H2AX constitute additional DDR signals, and BRCA1/BARD1 catalyzed K6-linked chains (not shown) may also contribute to DDR. Specific activities that oppose to these atypical ub chains in DDR are not known. 53BP1 and BRCA1 determine effective DSB repair, with 53BP1 committing to NHEJ (non-homologous end joining) and BRCA promoting HR (homologous recombination). Dashed lines indicate proposed protein–protein interactions. X indicates an unknown RNF8 substrate(s), and Y and W indicate unknown RNF168 substrates. Please refer to the main text for details.
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Figure 1: The role of deubiquitinating enzymes in the chromatin-based response to DNA double-strand breaks. (A) Recognition of a DNA double-strand break (DSB) by the MRN (MRE11-RAD50-NBS1) complex initiates DDR signaling, triggering ATM (ataxia-telangectasia mutated) kinase-dependent phosphorylation of H2AX (γH2AX). ATM phosphorylates also MDC1 (mediator of DNA damage checkpoint protein 1), which is recognized by the RING finger 8 (RNF8) E3 ligase. The activity of RNF8 is required for recruitment of a second E3, RNF168. RNF168 mono-ubiquitinates H2A-type histones on Lys13 and Lys15 (H2A(X)K13/K15Ub), and the concerted action of RNF8/RNF168 leads to the formation of K63-linked ubiquitin (Ub) chains on these lysines and to ubiquitination of other substrates (Y and W). RNF168 can bind to its own products, thereby amplifying chromatin ubiquitination around the DSB. OTUB1 opposes RNF168 activity in a non-catalytic manner, by binding to the E2 ubiquitin-conjugating enzymes UBC13 and UbcH5. USP3, USP44, and Dub3 DUB activities impair RNF168 recruitment, suggesting that they can target RNF8 substrate(s). These DUBs may also cleave RNF168-mediated ubiquitinated H2A(X). Excessive RNF168-dependent chromatin ubiquitination is limited by the TRIP12 and UBR5 E3 ligases, which target RNF168 for proteasomal degradation. USP34, instead, counteracts DSB-induced RNF168 ubiquitination. DSBs also trigger the recruitment of the Polycomb group E3 RING1B/BMI1, which mono-ubiquitinates H2A on Lys119 (H2AK119Ub) to locally repress transcription. USP16 and BAP1 target the H2AK119Ub mark, and USP16 activity is required for re-activation of DSB-induced transcriptional silencing. USP3 and USP44 oppose to steady-state mono-ubiquitinated H2A, which is primarily constituted by H2AK119Ub. (B) DSB-induced ubiquitin signals are recognized by downstream DDR factors. 53BP1 (p53 binding protein 1) recognizes the H2AK15Ub mark by its UDR (ubiquitination-dependent recruitment) motif, and dimethylated H4K20 (H4K20me2) with its TUDOR domain. One way BRCA1 (Breast cancer 1) is recruited to DSBs is through its interaction with RAP80 (receptor-associated protein 80), a protein that effectively binds K63-ub conjugates. BRCA1 has E3 ligase activity and functions as a heterodimer with BARD1 (BRCA1-associated RING domain protein 1). The DUBs BRCC36, POH1, and OTUB2 regulate DDR signaling by hydrolyzing DSB-induced K63-ub chains. OTUB2 also opposes to RNF8-dependent ubiquitination of L3MBTL1 (not shown). Whether MYSM1, which also possesses K63-ub cleavage activity, participates in this step of DDR is an open question. RNF168-mediated K27-linked ub chains on H2A/H2AX constitute additional DDR signals, and BRCA1/BARD1 catalyzed K6-linked chains (not shown) may also contribute to DDR. Specific activities that oppose to these atypical ub chains in DDR are not known. 53BP1 and BRCA1 determine effective DSB repair, with 53BP1 committing to NHEJ (non-homologous end joining) and BRCA promoting HR (homologous recombination). Dashed lines indicate proposed protein–protein interactions. X indicates an unknown RNF8 substrate(s), and Y and W indicate unknown RNF168 substrates. Please refer to the main text for details.

Mentions: In eukaryotic cells, the packaging of DNA with histone proteins into chromatin, the basic unit being the nucleosome, has major impact on DNA damage signaling and repair. This is because on one hand the compact organization of chromatin intrinsically limits the degree of access to DNA. At the same time, however, chromatin provides a sensitive regulatory platform for DDR through post-translational modifications to both chromatin components (i.e., histones) and non-chromatin proteins (Lukas et al., 2011). Non-proteolytic (mono-) ubiquitination of histones is a prevalent modification in mammalian cells (Goldknopf and Busch, 1977). Work from several groups has shown that histone ubiquitination at the chromatin surrounding DSBs is a key step in DDR activation (Lukas et al., 2011; Figure 1). Phosphorylation of the histone variant H2AX (yielding γH2AX) by the ataxia telangectasia mutated (ATM) checkpoint kinase promotes the binding of the E3 ligase RNF8 through the mediator protein MDC1 to damage sites, where it initiates ub signaling (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Wang and Elledge, 2007). A second E3, RNF168, is then recruited through RNF8-ubiquitinated substrates to the proximity of the lesion and starts catalysis of H2A/H2AX ubiquitination on Lys 13 (H2AK13ub) and/or Lys15 (H2AK15ub; Gatti et al., 2012; Mattiroli et al., 2012). The coordinated activities of RNF8/RNF168 with HERC2 and the E2 Ubc13 lead to the formation on H2A/H2AX of K63-ub, a prevalent ub linkage at DSBs (Doil et al., 2009; Pinato et al., 2009; Stewart et al., 2009; Gatti et al., 2012; Mattiroli et al., 2012). A major outcome of RNF8/RNF168-mediated ubiquitination is recruitment/stable accumulation of DDR proteins at the lesion, with the tumor suppressors BRCA1 (breast cancer 1, early onset) and 53BP1 (p53 binding protein 1) representing the two key effectors of the pathway (Lukas et al., 2011). Importantly, the interplay between BRCA1 and 53BP1 determines effective DSB repair by one of the two major DSB repair pathways, with BRCA1 promoting the error-free homologous recombination (HR) process while 53BP1 committing to non-homologous end joining (NHEJ; Cao et al., 2009; Bouwman et al., 2010; Bunting et al., 2010). By influencing the relative kinetics of these DDR effectors at DSBs, the RNF8 pathway is functionally implicated in determining the repair pathway choice, which is critical to genome maintenance (Panier and Boulton, 2014).


Fine-tuning the ubiquitin code at DNA double-strand breaks: deubiquitinating enzymes at work.

Citterio E - Front Genet (2015)

The role of deubiquitinating enzymes in the chromatin-based response to DNA double-strand breaks. (A) Recognition of a DNA double-strand break (DSB) by the MRN (MRE11-RAD50-NBS1) complex initiates DDR signaling, triggering ATM (ataxia-telangectasia mutated) kinase-dependent phosphorylation of H2AX (γH2AX). ATM phosphorylates also MDC1 (mediator of DNA damage checkpoint protein 1), which is recognized by the RING finger 8 (RNF8) E3 ligase. The activity of RNF8 is required for recruitment of a second E3, RNF168. RNF168 mono-ubiquitinates H2A-type histones on Lys13 and Lys15 (H2A(X)K13/K15Ub), and the concerted action of RNF8/RNF168 leads to the formation of K63-linked ubiquitin (Ub) chains on these lysines and to ubiquitination of other substrates (Y and W). RNF168 can bind to its own products, thereby amplifying chromatin ubiquitination around the DSB. OTUB1 opposes RNF168 activity in a non-catalytic manner, by binding to the E2 ubiquitin-conjugating enzymes UBC13 and UbcH5. USP3, USP44, and Dub3 DUB activities impair RNF168 recruitment, suggesting that they can target RNF8 substrate(s). These DUBs may also cleave RNF168-mediated ubiquitinated H2A(X). Excessive RNF168-dependent chromatin ubiquitination is limited by the TRIP12 and UBR5 E3 ligases, which target RNF168 for proteasomal degradation. USP34, instead, counteracts DSB-induced RNF168 ubiquitination. DSBs also trigger the recruitment of the Polycomb group E3 RING1B/BMI1, which mono-ubiquitinates H2A on Lys119 (H2AK119Ub) to locally repress transcription. USP16 and BAP1 target the H2AK119Ub mark, and USP16 activity is required for re-activation of DSB-induced transcriptional silencing. USP3 and USP44 oppose to steady-state mono-ubiquitinated H2A, which is primarily constituted by H2AK119Ub. (B) DSB-induced ubiquitin signals are recognized by downstream DDR factors. 53BP1 (p53 binding protein 1) recognizes the H2AK15Ub mark by its UDR (ubiquitination-dependent recruitment) motif, and dimethylated H4K20 (H4K20me2) with its TUDOR domain. One way BRCA1 (Breast cancer 1) is recruited to DSBs is through its interaction with RAP80 (receptor-associated protein 80), a protein that effectively binds K63-ub conjugates. BRCA1 has E3 ligase activity and functions as a heterodimer with BARD1 (BRCA1-associated RING domain protein 1). The DUBs BRCC36, POH1, and OTUB2 regulate DDR signaling by hydrolyzing DSB-induced K63-ub chains. OTUB2 also opposes to RNF8-dependent ubiquitination of L3MBTL1 (not shown). Whether MYSM1, which also possesses K63-ub cleavage activity, participates in this step of DDR is an open question. RNF168-mediated K27-linked ub chains on H2A/H2AX constitute additional DDR signals, and BRCA1/BARD1 catalyzed K6-linked chains (not shown) may also contribute to DDR. Specific activities that oppose to these atypical ub chains in DDR are not known. 53BP1 and BRCA1 determine effective DSB repair, with 53BP1 committing to NHEJ (non-homologous end joining) and BRCA promoting HR (homologous recombination). Dashed lines indicate proposed protein–protein interactions. X indicates an unknown RNF8 substrate(s), and Y and W indicate unknown RNF168 substrates. Please refer to the main text for details.
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Figure 1: The role of deubiquitinating enzymes in the chromatin-based response to DNA double-strand breaks. (A) Recognition of a DNA double-strand break (DSB) by the MRN (MRE11-RAD50-NBS1) complex initiates DDR signaling, triggering ATM (ataxia-telangectasia mutated) kinase-dependent phosphorylation of H2AX (γH2AX). ATM phosphorylates also MDC1 (mediator of DNA damage checkpoint protein 1), which is recognized by the RING finger 8 (RNF8) E3 ligase. The activity of RNF8 is required for recruitment of a second E3, RNF168. RNF168 mono-ubiquitinates H2A-type histones on Lys13 and Lys15 (H2A(X)K13/K15Ub), and the concerted action of RNF8/RNF168 leads to the formation of K63-linked ubiquitin (Ub) chains on these lysines and to ubiquitination of other substrates (Y and W). RNF168 can bind to its own products, thereby amplifying chromatin ubiquitination around the DSB. OTUB1 opposes RNF168 activity in a non-catalytic manner, by binding to the E2 ubiquitin-conjugating enzymes UBC13 and UbcH5. USP3, USP44, and Dub3 DUB activities impair RNF168 recruitment, suggesting that they can target RNF8 substrate(s). These DUBs may also cleave RNF168-mediated ubiquitinated H2A(X). Excessive RNF168-dependent chromatin ubiquitination is limited by the TRIP12 and UBR5 E3 ligases, which target RNF168 for proteasomal degradation. USP34, instead, counteracts DSB-induced RNF168 ubiquitination. DSBs also trigger the recruitment of the Polycomb group E3 RING1B/BMI1, which mono-ubiquitinates H2A on Lys119 (H2AK119Ub) to locally repress transcription. USP16 and BAP1 target the H2AK119Ub mark, and USP16 activity is required for re-activation of DSB-induced transcriptional silencing. USP3 and USP44 oppose to steady-state mono-ubiquitinated H2A, which is primarily constituted by H2AK119Ub. (B) DSB-induced ubiquitin signals are recognized by downstream DDR factors. 53BP1 (p53 binding protein 1) recognizes the H2AK15Ub mark by its UDR (ubiquitination-dependent recruitment) motif, and dimethylated H4K20 (H4K20me2) with its TUDOR domain. One way BRCA1 (Breast cancer 1) is recruited to DSBs is through its interaction with RAP80 (receptor-associated protein 80), a protein that effectively binds K63-ub conjugates. BRCA1 has E3 ligase activity and functions as a heterodimer with BARD1 (BRCA1-associated RING domain protein 1). The DUBs BRCC36, POH1, and OTUB2 regulate DDR signaling by hydrolyzing DSB-induced K63-ub chains. OTUB2 also opposes to RNF8-dependent ubiquitination of L3MBTL1 (not shown). Whether MYSM1, which also possesses K63-ub cleavage activity, participates in this step of DDR is an open question. RNF168-mediated K27-linked ub chains on H2A/H2AX constitute additional DDR signals, and BRCA1/BARD1 catalyzed K6-linked chains (not shown) may also contribute to DDR. Specific activities that oppose to these atypical ub chains in DDR are not known. 53BP1 and BRCA1 determine effective DSB repair, with 53BP1 committing to NHEJ (non-homologous end joining) and BRCA promoting HR (homologous recombination). Dashed lines indicate proposed protein–protein interactions. X indicates an unknown RNF8 substrate(s), and Y and W indicate unknown RNF168 substrates. Please refer to the main text for details.
Mentions: In eukaryotic cells, the packaging of DNA with histone proteins into chromatin, the basic unit being the nucleosome, has major impact on DNA damage signaling and repair. This is because on one hand the compact organization of chromatin intrinsically limits the degree of access to DNA. At the same time, however, chromatin provides a sensitive regulatory platform for DDR through post-translational modifications to both chromatin components (i.e., histones) and non-chromatin proteins (Lukas et al., 2011). Non-proteolytic (mono-) ubiquitination of histones is a prevalent modification in mammalian cells (Goldknopf and Busch, 1977). Work from several groups has shown that histone ubiquitination at the chromatin surrounding DSBs is a key step in DDR activation (Lukas et al., 2011; Figure 1). Phosphorylation of the histone variant H2AX (yielding γH2AX) by the ataxia telangectasia mutated (ATM) checkpoint kinase promotes the binding of the E3 ligase RNF8 through the mediator protein MDC1 to damage sites, where it initiates ub signaling (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Wang and Elledge, 2007). A second E3, RNF168, is then recruited through RNF8-ubiquitinated substrates to the proximity of the lesion and starts catalysis of H2A/H2AX ubiquitination on Lys 13 (H2AK13ub) and/or Lys15 (H2AK15ub; Gatti et al., 2012; Mattiroli et al., 2012). The coordinated activities of RNF8/RNF168 with HERC2 and the E2 Ubc13 lead to the formation on H2A/H2AX of K63-ub, a prevalent ub linkage at DSBs (Doil et al., 2009; Pinato et al., 2009; Stewart et al., 2009; Gatti et al., 2012; Mattiroli et al., 2012). A major outcome of RNF8/RNF168-mediated ubiquitination is recruitment/stable accumulation of DDR proteins at the lesion, with the tumor suppressors BRCA1 (breast cancer 1, early onset) and 53BP1 (p53 binding protein 1) representing the two key effectors of the pathway (Lukas et al., 2011). Importantly, the interplay between BRCA1 and 53BP1 determines effective DSB repair by one of the two major DSB repair pathways, with BRCA1 promoting the error-free homologous recombination (HR) process while 53BP1 committing to non-homologous end joining (NHEJ; Cao et al., 2009; Bouwman et al., 2010; Bunting et al., 2010). By influencing the relative kinetics of these DDR effectors at DSBs, the RNF8 pathway is functionally implicated in determining the repair pathway choice, which is critical to genome maintenance (Panier and Boulton, 2014).

Bottom Line: Signaling processes mediated by ubiquitin (ub) are crucial for the cellular response to DNA double-strand breaks (DSBs), one of the most dangerous types of DNA lesions.How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation.One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs), as supported by the implication of a growing number of DUBs in DNA damage response processes.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam Netherlands.

ABSTRACT
Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin (ub) are crucial for the cellular response to DNA double-strand breaks (DSBs), one of the most dangerous types of DNA lesions. In particular, the DSB response critically relies on active ubiquitination by the RNF8 and RNF168 ub ligases at the chromatin, which is essential for proper DSB signaling and repair. How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation. One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs), as supported by the implication of a growing number of DUBs in DNA damage response processes. Here, we discuss the current knowledge of how ub-mediated signaling at DSBs is controlled by DUBs, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer.

No MeSH data available.


Related in: MedlinePlus