5. RAD18: a key regulator of cell response to DNA damage
5.2. Recruitment of RAD18 to DNA damage sites
According to the polymerase switch model, translesion polymerases replace the replicative polymerases, and get access to chromatin at sites of DNA damage during the S-phase in order to allow the release of stalled forks (Friedberg et al., 2005). Therefore, PCNA monoubiquitination through RAD18-RAD6 must be tightly coordinated with the arrests of replication forks.
When the replicative polymerases are blocked by bulky DNA lesions, the helicase continues to unwind the DNA double-helix, giving long stretches of single-stranded DNA (ssDNA) that is coated and stabilized by the ssDNA bonding protein RPA (Byun et al., 2005).
Serval lines of evidence suggest that the RAD6-RAD18 complex may be recruited to stalled replication forks through interaction with this RPA-coated ssDNA. The first one is that RAD18 binds ssDNA through a DNA-interacting SAP domain that is necessary for recruitment of RAD18 to replication factories upon UV-irradiation (Byun et al., 2005; Nakajima et al., 2006;
Notenboom et al., 2007). Also, in vitro PCNAmonoUb by RAD18-RAD6 complex requires the loading of the PCNA clamp onto ssDNA (Garg and Burgers, 2005; Haracska et al., 2006). In addition, aphidicolin-induced replication fork uncoupling in Xenopus egg extracts leads to PCNAmonoUb (Chang et al., 2006). Interestingly, upon aphidicolin treatment, inhibition of replication fork uncoupling by blocking the helicase co-factor CDC45 using specific antibodies, prevents PCNAmonoUb (Chang et al., 2006). Finally, RPA interacts directly with RAD18 in vitro in budding yeast through an RPA interacting domain, and this interaction is required for PCNAmonoUb after MMS-induced DNA damage (Davies et al., 2008), whereas RAD18 chromatin binding is independent of RPA in Xenopus egg extracts (Recolin and Maiorano unpublished results).
Additionally, the MRN complex protein NBS1 has been shown to bind RAD18 after UV irradiation and mediate its recruitment to sites of DNA damage (Yanagihara et al., 2011).
NBS1 knock-down strongly reduces PCNAmonoUb and disrupts Pol η foci formation, giving an exacerbated UV sensitivity and elevated mutation rate (Yanagihara et al., 2011). The NBS1 binding domain was mapped on the C-terminal region of RAD18, and shares structural and functional similarities with the RAD6 binding domain. RAD18 homodimers can interact simultaneously with both NBS1 and RAD6. Therefore, NBS1 plays a role in translesion DNA synthesis in addition to its role in DSBs repair (Figure 5. 2) (Yanagihara et al., 2011).
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Figure 5. 2NBS1 role in translesion synthesis and homologous recombination (Yanagihara et al., 2011)
(A) NBS1 binds to E3 ubiquitin ligase RAD18 and targets it to site of UV lesions to mediate PCNA monoubiquitination and TLS-dependent lesion bypass. NBS1 binding domain on RAD18 shares structural similarity with RAD6 binding domain. (B) NBS1 plays also a role in DSBs repair within the MRN complex (see the text for more details).
Upon UV-induced DNA damage, RAD18 binds to DNA, but howRAD18 is targeted specifically to PCNA at stalled forks is poorly understood as RAD18 lacks PCNA-binding motifs. Few years ago, Spartan was identified as a scaffold for recruiting RAD18 to PCNAas it binds and bridges both RAD18 and PCNA (Centore et al., 2012). Consistent with this, DNA damage-induced PCNAmonoUb was partially reduced in Spartan-depleted cells (Centore et al., 2012). However, several other publications have suggested different roles for Spartan in DNA damage signalling such as protecting the ubiquitin residue on PCNA or recruiting the ubiquitin-selective chaperone p97 (Davis et al., 2012; Juhasz et al., 2012; Machida et al., 2012; Mosbech et al., 2012).
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In mammalian cells, RAD18 forms a complex with Polh (Figure 5. 3) (Day et al., 2010b; Watanabe et al., 2004), and this interaction is necessary for DNA damage tolerance (Barkley et al., 2012), as it targets RAD18 to PCNA and facilitates efficient PCNA monoUb. The S-phase checkpoint controls the RAD18–Pol h complex formation through CDC7 and CHK1 kinases (Day et al., 2010b; Watanabe et al., 2004). Surprisingly, Pol h stimulation of PCNAmonoUb is completely dissociable from its DNA polymerase activity. This may explain why catalytically inactive Pol h can partially rescue the DNA damage-sensitivity of XPV cells and induce the recruitment of other error-prone TLS polymerases (Pol k and Pol i) after UV irradiation (Ito et al., 2012b; Pavlov et al., 2001).
Figure 5. 3RAD18 simplified interactome
The diagram shows the main proteins that interacts with RAD18, and which play a role in the global cell response to DNA damage. In green background, proteins involved in translesion DNA synthesis.
Proteins involved in homologous recombination are depicted in purple background. Yellow background,
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template switch proteins. Orange background, DSBs signalling. Dark blue, regulatory protein. Light blue, non-homologous end joining protein. Red background, RAD18 ssDNA binding.
Recently, it has been shown that, upon DNA damage, c-Jun N-terminal Kinase (JNK) phosphorylates RAD18 specifically at S409 within the Polη binding domain, and in a checkpoint dependent manner. Interestingly, RAD18S409 phosphorylation promotes association with Polη (Barkley et al., 2012). Furthermore, the serine-threonine kinase CDC7 together with its S-phase regulator ASK (DBF4) phosphorylates a cluster of serine residues within the Polη binding domain of RAD18, and positively regulates Pol η–RAD18 interaction in an ATR-CHK1-mediated checkpoint dependent manner (Day et al., 2010b; Vaziri and Masai, 2010).
Upon replication fork block, CHK1 kinase inactivates the Anaphase-Promoting Complex/Cyclosome APC/C (Cdh1) through degradation of Cdh1, Thus APC/C (Cdh1) substrates are stabilized, including CDC7-ASK (DBF4) that phosphorylates RAD18 (Figure 5.
4). Moreover, the ASK (DBF4) subunit interacts with the N-terminal region of RAD18 through its motif-C, a conserved C2H2-type zinc finger domain (Figure 5. 4) (Yamada et al., 2013a).
This interaction is necessary for RAD18 chromatin binding and subsequent Polη foci formation (Yamada et al., 2013a).
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Figure 5. 4DDK-mediated recruitment of RAD18–Pol η complex upon replication stress (Yamada et al., 2014)
(A) Replication forks encounter a DNA lesion and stall. (B) Stalled forks activate the replication checkpoint, which inactivates the APC/CCdh1 pathway thus stabilizing DDK on chromatin. DDK phosphorylates RAD18 and promotes the interaction between RAD18 and Pol η, targeting RAD18 on site of damage where it monoubiquitinates PCNA, independently of DDK. (C) The DDK-RAD18-Pol η complex is loaded on chromatin to promote TLS. Within this complex RAD18 directly interacts with Dbf4.