Regulation of DNA Damage Responses by Ubiquitin and SUMO Stephen P. Jackson, Daniel Durocher  Molecular Cell  Volume 49, Issue 5, Pages 795-807 (March 2013) DOI: 10.1016/j.molcel.2013.01.017 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 The Ubiquitin and UBL Conjugation Cycle Ubiquitin and SUMO are produced as precursor polypeptides that are first processed to reveal a C-terminal diglycine motif (1). An E1 enzyme then uses ATP to convert this motif into a high-energy-bond containing adenylated derivative, which is short-lived, rapidly reacting with a Cys in the E1 to form a E1∼Ub or E1∼SUMO thioester intermediate (2). The ubiquitin or SUMO moieties are then coupled, via a transesterification reaction, onto a Cys residue in the E2 catalytic site to form E2∼Ub/SUMO intermediates (3). In most cases, an E3 ligase serves as a substrate adaptor, linking the charged E2 and the substrate (4). Ubiquitylation or sumoylation can be reversed by a DUB (for ubiquitin) or a SENP (for SUMO) (5) or the modification cycle can be repeated to produce chains of various topologies (6). The (∗) indicates that other types of enzymes can remove UBL modifications. Molecular Cell 2013 49, 795-807DOI: (10.1016/j.molcel.2013.01.017) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 The roles of Ubiquitin and SUMO in Postreplication Repair (A) PCNA monoubiquitylation on Lys164 occurs when replication forks encounter lesions (star). The single-stranded DNA is recognized by the Rad18 E3 ligase, which interacts with Rad6, an E2, to ubiquitylate PCNA. PCNA ubiquitylation recruits Y family polymerases such as Polη to bypass the lesion in the process termed translesion synthesis (TLS). In vertebrates the USP1-UAF1 DUB opposes PCNA monoubiquitylation. (B) A second DNA damage tolerance pathway termed template switching occurs when PCNA is polyubiquitylated by the E3 Rad5 and the Ubc13/Mms2 dimeric E2. Template switching employs homologous recombination. (C) In yeast, PCNA sumoylation on its Lys164 residue recruits Srs2, which acts as an antirecombinase by disrupting Rad51 nucleoprotein filaments. Molecular Cell 2013 49, 795-807DOI: (10.1016/j.molcel.2013.01.017) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 The Role of Ubiquitin in Nucleotide Excision Repair (A) GG-NER promotes the repair of bulky lesions on genomic DNA. The lesions can be recognized by the DDB1-DDB2 and XPC-RAD23 complexes. DDB1-DDB2 forms an E3 with CUL4 and RBX1 (not shown), which leads to DDB2 autoubiquitylation, resulting in its degradation, and XPC polyubiquitylation. XPC is stabilized through its interaction with RAD23, which contains a ubiquitin-like domain that interacts with the proteasome. This allows the subsequent steps of NER, such as the recruitment of XPA. (B) TC-NER repairs bulky lesions occurring on the transcribed DNA strand. RNA polymerase II acts as a lesion sensor. Two independent pathways can occur when RNA polymerase stalls after a lesion encounter. First (going right), TC-NER can be activated, with the CSB and CSA proteins playing a critical role in TC-NER. CSB associates with RNA polymerase and recruits CSA, which forms a CUL4-based E3 with DDB1. The exact nature of the critical substrate of the E3 associated with CSA is not known, but CSB is ubiquitylated and its degradation is delayed by USP7, which is brought to the stalled polymerase by UVSSA. Second (going down), the RNA polymerase can be ubiquitylated, extracted from chromatin, and degraded by the proteasome as a last resort. This pathway, in yeast, is initiated by Rad26-Def1 (not shown), which leads to the Rsp5-dependent ubiquitylation of RNA polymerase. Rsp5 can promote Lys63-ubiquitin chains but the DUB Ubp2 trims these down. Monoubiquitylated RNA polymerase is then a substrate of an Elongin-Cullin 3 complex. The Lys48-linked ubiquitin chains on RNA polymerase enable Cdc48 to extract the stalled polymerase from chromatin, which then leads to its degradation. Molecular Cell 2013 49, 795-807DOI: (10.1016/j.molcel.2013.01.017) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 The Role of Ubiquitin in the Chromatin-Based Response to DNA Double-Strand Breaks (A) DSBs trigger the ATM-dependent phosphorylation of H2AX, which is read by MDC1. ATM can also phosphorylate MDC1 to promote the recruitment of RNF8, an E3 ligase. RNF8 ubiquitylates an unknown factor on chromatin (X). This ubiquitylation is then read by the N-terminal ubiquitin binding domains of RNF168. RNF168 ubiquitylates H2A, which provides a second recruitment signal for RNF168. In addition to RNF8/168, DSBs stimulate the recruitment of the BMI1-RING1B, which ubiquitylates H2A on the C terminus. Also shown are DUBs that antagonize this ubiquitylation event. (B) OTUB1 antagonizes RNF168-dependent ubiquitylation by binding to E2s. RNF8 and RNF168 also stimulate the formation of Lys63-linked ubiquitin chains on chromatin. (C) The RNF168-dependent ubiquitylation of chromatin leads to the recruitment of multiple effectors that include 53BP1, which promotes NHEJ, and BRCA1, which promotes HR. Molecular Cell 2013 49, 795-807DOI: (10.1016/j.molcel.2013.01.017) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 The Role of Ubiquitin in the FA Pathway ICLs are potent fork-blocking lesions. DNA replication fork stalling, along with FANCM and associated proteins (not shown), stimulates the recruitment of the multi-subunit FA core complex, which is an E3 ligase. The catalytic subunit of this E3 is FANCL and its E2 is UBE2T. The FA core complex monoubiquitylates FANCD2-FANCI. FANCD2-FANCI ubiquitylation is read by FAN1 and perhaps SLX4, which both contain a UBZ4-type ubiquitin binding domain. The USP1-UAF1 DUB reverses FANCD2-FANCI ubiquitylation and is essential for the FA pathway. The recruitment of nucleases enables incision, unhooking of the lesion, TLS, and HR. Molecular Cell 2013 49, 795-807DOI: (10.1016/j.molcel.2013.01.017) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Examples of the Role of SUMO in Multiple DNA Damage Response Pathways (A) Thymine-DNA glycosylase (TDG), which acts during BER, is inhibited by the product of its reaction. TDG sumoylation produces a conformational change that enables it to leave DNA. (B) Sumoylation acts at multiple steps during the chromatin-based DSB response. See the main text for details. (C) Sumoylation can also stimulate ubiquitylation through the action of STUbLs, E3 ligases that are recruited to substrates by recognizing sumoylation. In the shown example, RNF4 promotes the turnover of MDC1 from chromatin during the DSB response. Molecular Cell 2013 49, 795-807DOI: (10.1016/j.molcel.2013.01.017) Copyright © 2013 Elsevier Inc. Terms and Conditions