Genome integrity and stability, which are constantly threatened by a wide range of endogenous and exogenous DNA-damaging agents (Groelly et al., 2023), are essential for the living cell. Persistent DNA damage during the S phase can block replicative polymerases, leading to replication stalling, fork collapse, single-stranded gaps, double-stranded breaks and, consequently, gross chromosomal rearrangement (Ma et al., 2020, Ler and Carty, 2022). Stabilizing and restoring the stalled replication fork and filling the DNA discontinuities ensures the completion of replication and the maintenance of genomic integrity (Buoninfante et al., 2023).

Highly conserved DNA damage tolerance pathways (DDT) are involved in avoiding the fatal consequences that arise from the inhibition of DNA replication (Branzei and Szakal, 2016). The two main extensively researched DDT strategies are translesion synthesis (TLS) – which employs specialized TLS polymerases to bypass lesions at the site of replication arrest – and template switching (TS), during which the newly synthesized DNA strand of the sister chromatid serves as a template to rescue the stalled replication fork (Prakash et al., 2005) (Wang et al., 2016, Haracska et al., 2006, Unk et al., 2008) (Fig. 1A upper panel). Recent research has shown that cells utilize an additional mechanism called repriming to ensure the progression of stalled replication forks (Guilliam and Doherty, 2017, Quinet et al., 2021). One of the main players and drivers of this mechanism is the active primase PRIMPOL, whose ability to de novo synthesize DNA primers enables it to restart replication downstream of the DNA damage (Mourón et al., 2013, García-Gómez et al., 2013, Bianchi et al., 2013).

TS and TLS can occur in the S phase immediately when the replication fork stalls as well as in the later phases of the cell cycle (G2/M) to fill the remaining single-stranded gaps (this gap filling is also referred to as postreplicative repair) (Fig. 1B). In addition, the RAD51 homologous recombination pathway can also rescue replication stalling and fill the single-stranded DNA gaps formed during the replication of the damaged DNA (Piberger et al., 2020).

As replication forks stall upon encountering DNA damage, HRAD6A/B/RAD18 catalyzes the monoubiquitylation of PCNA, which results in the recruitment of TLS polymerases. PCNA can be further polyubiquitylated by MMS2-Ubc13 (a ubiquitin conjugase E2 enzyme complex) with HLTF or SHPRH (as a E3 ubiquitin ligase); this modification is the driving signal for template switching by various mechanisms such as fork reversal. Besides its central role in TLS and TS, RAD18 crosstalks with other DNA damage tolerance pathways such as homologous recombination (HR), the Fanconi anemia (FA) pathway, non-homologous end joining (NHEJ), and break-induced replication (BIR). Regulation of HR by RAD18 can be direct or indirect; the RAD18-dependent enhancement of the FA pathway is stimulated mostly through the promotion of ubiquitylation of FANCD2. RAD18 also monoubiquitylates 53BP1 and enhances its retention at double-stranded break sites, thereby directing the pathway choice between HR and the NHEJ. During break-induced telomer synthesis, RAD18-directed PCNA ubiquitylation recruits SNM1A to breaks. SNM1A promotes ssDNA generation to mediate a template switching-dependent lesion bypass at difficult-to-replicate regions such as telomeric secondary structures (for more detailed description of these pathways, see 1.1 TLS is directed by PCNA monoubiquitylation, 1.2 PCNA polyubiquitylation directs template switching/fork reversal, 1.3 RAD18 controls HR by direct protein interactions and through indirect signaling, 1.4 RAD18-mediated TLS activity is connected to the Fanconi anemia pathway, 1.5 RAD18 cross-talks with non-homologous end joining repair, 1.6 RAD18-dependent PCNA ubiquitylation signaling directs break-induced telomeric synthesis).

(B) Formation and filling in of single-stranded DNA gaps during replication stress

In the presence of a lesion, the replicative polymerase is arrested at the damage and, since the helicase can move forward, the DNA synthesis on the leading and the lagging strand can be uncoupled. Fork-associated bypass of the lesion can be accomplished immediately, e.g., by TLS, or the lesion can be skipped when PRIMPOL re-initiates the replication, leaving a gap behind. Gap filling can be postponed until later phases of the cell cycle (G2/M) (Bergoglio et al., 2013).

DNA distortions or base modifications lead to inhibited or blocked replication due to the arrest of the high-fidelity replicative DNA polymerases Polymerase delta (Polδ) and epsilon and Polε (Lujan et al., 2017). Replication stress involves mechanisms that disturb the progress of the replication fork or inhibit the metabolism and synthesis of nascent DNA. Replication stress, which is a hallmark of cancer, induces DNA damage response cascades and may lead to aberrant cell cycle regulation, oncogene activation, sustained proliferation, and genomic instability (Macheret and Halazonetis, 2015).

In the presence of a replication-blocking lesion, the progress of the replicative polymerase is halted at the damage site, but the replicative CMG helicase can still move forward, resulting in the uncoupling of the DNA synthesis on the leading and lagging strands, thus, in the accumulation of long stretches of ssDNA (Lopes and Foiani, 2006). The ssDNA at the fork, if left unprotected, is prone to nuclease attacks and can form non-preferable secondary structures, which pose barriers to transcription and replication; thus, the formation of ssDNA immediately induces the accumulation of RPA filaments at the stalled fork, which serve to shield and protect ssDNA (see also in Section 2.5) (Bhat and Cortez, 2018).

To overcome replication stress induced by replication barriers caused by to endogenous or exogenous factors, the replicative polymerase is temporarily replaced by TLS polymerases in a process directed by HRAD6A and HRAD6B (HRAD6A/B). These two human orthologs of the yeast RAD6 E2 ubiquitin-conjugating enzyme form a complex with the RAD18 E3 ubiquitin ligase (Fig. 1A upper panel). The HRAD6A/B/RAD18 complex monoubiquitylates proliferating cell nuclear antigen (PCNA) at lysin 164 as a response to DNA damage caused by UV, MMS, or cisplatin and various other types of agents (Moldovan et al., 2007, Hoege et al., 2002, Tateishi et al., 2003, Miyase et al., 2005, Watanabe et al., 2009a, Yoon et al., 2012, Prakash et al., 2005, Waters et al., 2009, Kannouche and Lehmann, 2006, Ripley et al., 2020, Bailly et al., 1994, Bailly et al., 1997).

The sliding clamp PCNA, which is a homotrimer protein belonging to the evolutionarily well-conserved β-clamp family (González-Magaña and Blanco, 2020, Fukuda et al., 1995) serves as a crucial loading platform for the physical anchoring of polymerases to DNA (Acharya et al., 2007, Prakash et al., 2005, Mailand et al., 2013, Haracska et al., 2006).

Monoubiquitylation of PCNA stabilizes the interaction of PCNA with TLS polymerases and enhances their further recruitment to the damage site, indicating that it is a crucial step in the activation of TLS (Maiorano et al., 2021). The details of the sequential actions of TLS, namely, the recruitment of RAD18 and TLS polymerases to the damage site and the mechanism of polymerase exchange are still not fully understood, despite the identification of new interactive partners of RAD18 and TLS polymerases that act as readers and/or enhancers of PCNA ubiquitylation.

RAD18 was shown to have some affinity to ssDNA (Bailly et al., 1997), but, according to several studies, the accumulation of RPA at the damage site contributes further to directing RAD18 to ssDNA (Davies et al., 2008, Press et al., 2019). After the recruitment of RPA, the HRAD6A/B/RAD18 complex randomly translocates along the RPA filaments, and this process increases the interaction of the HRAD6A/B/RAD18 complex with PCNA, thereby significantly enhancing PCNA monoubiquitylation (M. Li et al., 2021).

Importantly, RAD18 itself does not have a PCNA-interacting domain; therefore, its recruitment to PCNA requires accessory proteins. Multiple new interacting partners of RAD18 have been identified recently that could enhance its localization to PCNA and enable task-specific crosstalk with other DNA repair pathways, chromatin remodeling, and cell cycle regulation, emphasizing the central role of RAD18 in genome stabilization (Maiorano et al., 2021).

RAD18 and Polη form a complex in mammalian cells (Day et al., 2010, Watanabe et al., 2004), and this interaction plays a key role in targeting RAD18 to PCNA. The TLS polymerases Polη and Polκ have a so called PCNA-interacting PIP box, which enables interaction with PCNA. Durando et al. have found that the recruitment of RAD18 to PCNA strongly depends on Polη but not on Polκ (Durando et al., 2013). Polη acts as a specific scaffold to facilitate RAD18-PCNA interaction, and its C-terminus physically binds RAD18 to PCNA. This TLS regulatory function is independent of the polymerase activity of Polη and represents not a linear but rather a multilayer activation cascade, since RAD18 is not a simple, linear upstream activator of Polη, but these two proteins play mutually dependent roles in the activation of TLS (Durando et al., 2013).

The importance of the multilayer activation process of RAD18 and Polη is also reflected by the posttranslational modifications of Rad18 and Pol η, which have TLS modulatory roles.

It has been reported that Cdc7-dependent phosphorylation of RAD18 promotes efficient recruitment of Polη to stalled replication forks after UV treatment (Day et al., 2010, Barkley et al., 2012). Barkley et al. have found that JNK (c-Jun N-terminal kinase), a member of the stress-activated protein kinase (SAPK) family, phosphorylates RAD18 at S409, within the Polη-binding motif, which specifically promotes Rad18–Polη association, facilitates efficient recruitment of Polη to the replication machinery, and confers DNA damage tolerance (Barkley et al., 2012).

RAD18, independently of its ubiquitin ligase activity, promotes Polη SUMOylation on lysine K163 by facilitating its interaction with its SUMO ligase PIAS1. SUMOylated Polη was found to travel along the replication fork and detect lesion-independent replication stress at difficult-to-replicate DNA loci (common fragile sites, CFS). This RAD18-dependent SUMOylation of Polη facilitates the replication of these regions, thereby decreasing the intrinsic replication stress of these genomic loci and preventing the formation and persistence of ssDNA gaps, which could lead to under-replicated regions at CFS (Despras et al., 2016).

Besides TLS polymerases such as Polη and REV1 (Wang et al., 2016), numerous other interacting proteins have been reported to stimulate targeting of RAD18 to PCNA, possibly further specifying their interaction.

SIVA1 (Han et al., 2014) was found to serve as a molecular bridge between RAD18 and PCNA, and the RAD18 regions that mediate binding to SIVA overlap with the HRAD6A/B binding region (Han et al., 2014).

Claspin, a part of the fork protection complex, was found to be important for PCNA monoubiquitylation by coordinating the replicative helicase with PCNA and mediating the interaction between PCNA and RAD18 (Yang et al., 2008).

NBS1, one of the first responders to double-stranded breaks, physically binds and recruits RAD18 to the damage site (Yanagihara et al., 2011, Komatsu, 2016).

RFWD3, an E3 ubiquitin ligase, was reported to enhance PCNA monoubiquitylation by ubiquitylating multiple proteins on the RPA-coated ssDNA, thereby promoting ubiquitin-dependent accumulation of DDT proteins and stimulating bypass at DNA gaps (Gallina et al., 2021).

Chromatin remodeling factors such as ZBTB1 (Zinc finger and BTB domain containing 1) and SART3 (Squamous Cell Carcinoma Antigen Recognized By T-Cells 3) were found to increase TLS after UV irradiation. Recruitment of ZBTB1 (Kim et al., 2014) to sites of UV-induced cyclobutene pyrimidine dimers enables local chromatin relaxation by pKAP-1, which allows for the recruitment of RAD18, thus facilitating PCNA monoubiquitylation. SART3 can function as a histone chaperon and facilitate ssDNA generation followed by the promotion of RPA and RAD18 recruitment. It enhances RAD18/Polη interaction, stimulating TLS (Huang et al., 2018).

Spartan (Machida et al., 2012, Kim et al., 2013, Mosbech et al., 2012), the first identified replication-coupled DNA-protein crosslink repair protease (Mórocz et al., 2017, Lessel et al., 2014, Stingele et al., 2015, Reinking et al., 2020, Stingele et al., 2016, Vaz et al., 2017), was found to enhance PCNA monoubiquitylation. Spartan, on the one hand, is a reader of ubiquitylated PCNA and facilitates the access of Pol η to replication forks (Juhasz et al., 2012), on the other hand, by enhancing the association of RAD18 with chromatin, acts together with it in a feed-forward loop, thereby further increasing PCNA monoubiquitylation (Centore et al., 2012). The interaction of Spartan with monoubiquitylated PCNA protects against PCNA deubiquitylation by USP1 (Ubiquitin-specific protease 1) (Juhasz et al., 2012).

BRCA1, a crucial factor of homologous recombination (Moynahan et al., 1999), was also found to play a role in the promotion of the mono and polyubiquitylation of PCNA. BRCA1 regulates the recruitment of RPA, RAD18, and HLTF (helicase-like transcription factor) to the chromatin but also directly recruits TLS polymerases such as Polη and Rev1 to the lesions through protein-protein interaction (Tian et al., 2013b).

MAGE-A4 (melanoma antigen-A4), a neomorphic cancer cell-specific cancer/testes antigen (CTA), which is aberrantly expressed in many cancers, was shown to stabilize RAD18 and confer increased PCNA monoubiquitylation and TLS. RAD18 overexpression could enhance DNA damage tolerance (Kermi et al., 2015); thus, the MAGE-A4-RAD18 complex, as a mutagenic driver, could increase TLS capacity, contributing to replication stress tolerance and facilitating neoplastic cell survival and tumor progression (Gao et al., 2016). MAGE-A4 and RAD18-directed TLS enhancement may lead to genome destabilization and increased tolerance not only to endogenous oncogenic stress but also to chemo and radiotherapy, thus being a potential drug target.

Monoubiquitylation of PCNA on the K164 residue directs TLS polymerases to the stalled replication fork (Yoon et al., 2012, Prakash et al., 2005, Waters et al., 2009, Kannouche and Lehmann, 2006, Ripley et al., 2020, Bailly et al., 1994, Bailly et al., 1997) and enables replication to proceed through damaged DNA. Monoubiquitylated PCNA may induce the dissociation of replicative polymerases and enhance association with damage bypass polymerases (Kanao and Masutani, 2017a).

Monoubiquitylated PCNA preferentially interacts with the Y-family DNA polymerases via their ubiquitin-binding domains: UBZ (ubiquitin-binding zinc finger) in Polη (Johnson et al., 1999, Kannouche et al., 2004, Bienko et al., 2005) and Polymerase Kappa (Polκ) ((Stern et al., 2019) and UBM (ubiquitin-binding motif) in Polymerase iota (Polι) (Mcintyre, 2020, Mcintyre et al., 2013, Plosky et al., 2006) and REV1 (Wood et al., 2007, Kanao and Masutani, 2017a). In addition to UBZ/UBMs, Polη, (Acharya et al., 2007, Kannouche and Lehmann, 2006, Ohmori et al., 2009), Polι (Vidal et al., 2004, Mcintyre, 2020) and Polκ (Stern et al., 2019, Yoon et al., 2012) contain PIP (PCNA-interacting peptide) domains, and REV1 harbors a BRCT domain (Guo et al., 2006), which serves to interact with PCNA.

In addition to the interactions between TLS polymerases and monoubiquitylated PCNA, the mutual interactions among TLS polymerases also enhance their recruitment and activity (Kanao and Masutani, 2017b). REV1 acts as a platform for other TLS polymerases and potentially coordinates their activity by providing a scaffold to facilitate polymerase switching at the damage site (Guo et al., 2009, Wang et al., 2016).

Following the recruitment to the damage site, TLS polymerases – which have more flexible active sites than replicative polymerases, without associated proofreading activity – perform the replicative bypass of the lesion, which leads to the accumulation of mutations. However, Polη is able to perform error-free bypass of UV-induced CPD (cyclobutane-pyrimidine-dimers) lesions; in the absence of Polη, other low-fidelity polymerases bypass CPDs with mutagenic consequences, most likely Pol ι (Mansilla et al., 2023), Polκ, and the B-family Polymerase Zeta (Polζ) (Makarova and Burgers, 2015). Polζ consists of a catalytic subunit (Rev3) and a regulatory subunit (Rev7), the latter one interacting with REV1 (Gan et al., 2008).

TLS across a particular DNA lesion regularly involves two but may require as many as four different TLS polymerases in a sequential action of insertion and extension. Insertion is regularly error prone and determines the accuracy and mutagenic specificity of the TLS reaction and is carried out by Polη, Polκ, or Polι. In contrast, extension is carried out primarily by polζ (Livneh et al., 2010).

PCNA ubiquitylation-directed TLS, besides replication-coupled damage bypass, has an important role in the filling in of postreplicational gaps formed after repriming by PRIMPOL (Mourón et al., 2013, García-Gómez et al., 2013, Bianchi et al., 2013).

Monoubiquitylated PCNA can then be polyubiquitylated at the same K164 residue by the E2 enzyme Mms2-Ubc13 and the E3 ubiquitin ligases HLTF or SHPRH (Yathish Jagadheesh Achar et al., 2015; Unk et al., 2010). Polyubiquitylation of PCNA is crucial for directing the DDT pathway choice and activating the error-free damage tolerance pathway (Ripley et al., 2020, Ulrich and Jentsch, 2000, Ulrich, 2011). This K63-linked polyubiquitylation is associated with template switching (TS) (Fig. 1A, upper panel) (Achar et al., 2011; Y.J. Achar et al., 2015; Burkovics et al., 2014).

Template switching refers to molecular events in which the synthesis of DNA is started using one of the DNA strands as a template and then continued by copying the other strand, and there is still an ongoing debate about its most frequent mechanisms (Kondratick et al., 2021). Recently, fork reversal – in which the formation of a four-way junction structure involves the annealing of parental DNA strands, followed by the binding of daughter strands and thus the formation of a “chicken foot”-like structure – has come into the focus of interest (Blastyák et al., 2010, Unk et al., 2010). There are numerous fork remodelers that can reverse the stalled replication fork, including HLTF (Blastyák et al., 2010, Ciccia et al., 2012), SHPRH (Unk et al., 2006), ZRANB3 (Vujanovic et al., 2017), RAD51 (Zellweger et al., 2015), PARP1 (Ray Chaudhuri et al., 2012), FANCM (Gari et al., 2008), FBH1 (Fugger et al., 2015), BLM (Ralf et al., 2006), SMARCAL1 (Bétous et al., 2013), and WRN (Machwe et al., 2007).

There are several important processes through which fork reversal can affect genomic stability (Bhat and Cortez, 2018). First, fork regression can slow replication until a converging fork from a nearby origin replicates the region. Second, formation of the regressed fork places the lesion on the template strand back into the duplex DNA region where other repair mechanisms such as excision repair can process it. Third, annealing of the nascent strand can provide an undamaged template to synthesize past the lesion using the template switch mechanism. Finally, the reversed fork can promote recombination-mediated repair; it can be cleaved by structure-selective endonucleases such as MUS81 and SLX4. However, without further protection by BRCA1/2 (Schlacher et al., 2011), RAD51 (Schlacher et al., 2012), WRNIP (Leuzzi et al., 2016), FANCD2 (Yang et al., 2015a), REV1 (Yang et al., 2015a), WRN (Su et al., 2014), BODIL1 (Higgs et al., 2015), and RECQL5 (Kim et al., 2015), the reversed fork is susceptible to undesirable nucleolytic cleavage by exonucleases such as MRE11 (Kolinjivadi et al., 2017), EXO1 (Cotta-Ramusino et al., 2005), and DNA2 (Thangavel et al., 2015). The restoration of fork stability and protection is a way of developing chemoresistance in BRCA1/BRCA2-depleted tumors; thus, participants of this process may be important cancer chemotherapy targets (Taglialatela et al., 2017, Bai et al., 2020).

Double-stranded breaks (DSBs) are the primary cytotoxic lesions in a living cell arising from ionizing radiation, mechanical stress on chromosomes, radio-mimetic drugs, or topoisomerase poison chemicals such as camptothecin (CPT) (Jasin and Rothstein, 2023, Tripathi et al., 2016); thus, if left unrepaired or are repaired incorrectly, they can give rise to mutations and chromosomal instability. The two primary pathways for repairing double-stranded breaks in mammalian cells are HR and non-homologous end joining (NHEJ) (see also Section 1.5) (Scully et al., 2020). HR ensures the precise repair of double-stranded breaks through the utilization of an undamaged DNA strand from an intact sister chromatid as a template for DNA polymerase extension across the break. This process is restricted to the late S and G2 phases of the cell cycle.

Szüts et al. (Szüts et al., 2006) have found that RAD18 has a direct role in homologous recombination and detected reduced efficiency of gene conversion (induced by targeted double-stranded breaks) in a reporter construct in rad18 chicken DT40 cells, concluding that homologous recombination is toxic in the absence of RAD18 and leads to increased numbers of aberrant rearrangements at their immunoglobulin loci. Moreover, Huang et al. found that RAD18 is recruited to double-stranded break sites via its zinc finger domain in a RNF8/UBC13-dependent manner (Huang et al., 2009, Kobayashi et al., 2015), and the function of RAD18 in HR is not dependent on either its E3 ligase activity or its interaction with RAD6.

During homologous recombination, RAD18 also acts as an adaptor protein by directly binding to RAD51C, thus enabling the accumulation of RAD51C at the sites of DNA damage, promoting the formation of Rad51 foci, and enhancing HR repair. This suggests that RAD18 transmits DNA damage signals from the pathway involving ATM, MDC1, and RNF8 to facilitate DSB repair via direct interaction with RAD51C in HR (Huang et al., 2009, Ting et al., 2010).

RAD18 was also reported to be an important signaling factor at DSBs following ionizing irradiation exposure, since, in its absence, DNA damage signaling and checkpoint activation was attenuated, and ATM, γH2AX, and 53BP1 foci formation was decreased at the G2/M phase (Sasatani et al., 2015). Successful repair of double-stranded breaks may require the joint operation of several repair pathways. For complete HR-directed removal of replication-associated DSBs induced by CPT, functional interaction of BRCA2, FANCD2, Rad18, and Rad51 is required, since loss of any of these proteins leads to equal disruption of HR repair (Tripathi et al., 2016).

Fanconi anemia (FA) is a rare genetic disorder associated with cancer susceptibility, bone marrow failure, congenital anomalies and characterized by chromosomal abnormality and genomic instability (Peake and Noguchi, 2022, Liu et al., 2020). The disease is caused by loss of function of any one of the 22 known FA genes (Rageul and Kim, 2020).

The FA pathway plays a critical role in repairing DNA damage and rescuing replication, particularly in the presence of interstrand crosslinks (ICLs) (Q. Li et al., 2021). The activity of this multiplayer pathway involves three main complexes: the Core Complex (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM), the ID2 Complex (FANCI, FANCD2), and the downstream effector complex (BRCA2, FANCN/PALB2, BRCA1, FANCJ, FANCO, and FANCR) (Gianni et al., 2022). Several Fanconi anemia pathway members operate in other DDT pathways, e.g., the tumor suppressor proteins BRCA1 (also named as FANCS) and BRCA2 (also named as FANCD1), which, besides their primary roles in double-stranded DNA break repair, also control replication, transcription, and cell cycle (Gianni et al., 2022), and BRCA1 has a RAD18-connected TLS stimulatory activity, reflecting multiple crosstalks between FA, TLS and TS pathways (Tian et al., 2013a).

A crucial event in the activation of the FA pathway is represented by the monoubiquitylation of FANCD2 and FANCI in response to DNA damage, which involves the monoubiquitylation of FANCD2 by the E3 ligase FANCL. It has been demonstrated that in the absence of RAD18 the activation of the FA pathway is attenuated, and, correspondingly, the damage-induced mono-ubiquitylation of both PCNA and FANCD2 is attenuated. The fact that E3 ligase-deficient RAD18 mutant was not able to fully restore PCNA ubiquitylation and FANCD2 activity, strongly supports that the activation of FANCD2 requires PCNA ubiquitylation. RAD18-directed PCNA monoubiquitylation promotes FA core complex-dependent FANCD2 monoubiquitylation, but RAD18 itself is insufficient to fully activate it (Song et al., 2010). RAD18 was shown to stimulate FANCL-catalyzed ubiquitylation of both FANCD2 and FANCI through the monoubiquitylation of PCNA. Codepletion of RAD18 and FANCD2 did not result in additive sensitization to cisplatin, which raised the possibility that these proteins might act in the same pathway (Geng et al., 2010) (Williams et al., 2011).

Although the FA pathway primarily operates in the repair of ICLs, it has also been associated with response to challenges posed by transcription-replication conflicts and R-loops (García-Rubio et al., 2015, Schwab et al., 2015). In a recently published study, Wells et al. demonstrated that loss of RAD18 leads to the accumulation of DNA:RNA hybrids because RAD18-depleted cells have higher levels of transcription-replication conflicts. In the absence of RAD18, the failure of FANCD2 recruitment at difficult-to-replicate and R-loop prone genomic sites induces replication stress and leads to DSB formation. They suggested that RAD18-mediated PCNA ubiquitylation is implicated in R-loop accumulation, since the physical loss of RAD18 by depletion does not further enhance R-loops in PCNA K164R mutant cells (Wells et al., 2022).

The pathways choice between the nonhomologous end joining (NHEJ) and homologous recombination (HR) (Symington and Gautier, 2011) depends on the phase of the cell cycle and the nature of the DSB ends. Some NHEJ factors, such as 53BP1, promote direct joining of DSBs by protecting the DNA ends from resection, which is needed for HR initiation (Ghezraoui et al., 2020). RAD18 associates with 53BP1 and is recruited to DSBs in a 53BP1-dependent manner, particularly during the G1 phase (Watanabe et al., 2009b). Moreover, RAD18 monoubiquitylates 53BP1, which was demonstrated to play a functional role in promoting the retention of 53BP1 at DSB sites.

RAD18 was reported to be an important regulator of the level of 53BP1, since RAD18 knockout led to the increase of 53BP1 (Nieto et al., 2020), suggesting that RAD18 promotes degradation of 53BP1. This function may be important in making the choice between the HR and NHEJ pathways, which is governed by the opposing roles of 53BP1 and BRCA1. According to a recent model, the DSB repair pathway choice is controlled by a cell cycle-regulated inhibition of 53BP1-RIF1 and BRCA1-CtIP: the former is dominant in G1, and the latter is the regulator in S/G2 (Escribano-Díaz et al., 2013). In a variety of malignancies, RAD18 has been found to be aberrantly expressed, which can promote TLS and DSB repair, leading to increased resistance to replication stress and DNA damage, thus contributing to cancer development and progression (Huang et al., 2009, Yang et al., 2018, Baatar et al., 2020, Xie et al., 2014, Li et al., 2022).

RAD18 has been reported to direct BITS (break induced telome synthesis), which is a form of break induced replication (BIR), and contributes to alternative lengthening of telomeres, independently of RAD51. BITS executes conservative, unidirectional, long-track homologous replication repair DNA synthesis using minimal replisome, containing PCNA and Polymerase- delta (Zhang et al., 2023). Break-induced replication is a highly error-prone process associated with frequent template switches (Dilley et al., 2016, Malkova, 2018), and its mechanism of replicating through difficult-to-replicate regions such as telomeric secondary structures was largely unknown. Zhang and colleagues reported that replication stress

during break-induced telomer synthesis induces RAD18-directed PCNA ubiquitylation, which recruits SNM1A to breaks. SNM1A, an 5’-3’ exonuclease, has a role connected to DNA interstrand cross link repair (Baddock et al., 2020, Buzon et al., 2018). SNM1A executes endonuclease-mediated DNA nicking and 5′–3′ exonuclease resection in break-induced telomer synthesis, promoting ssDNA generation to mediate a template switching-dependent lesion bypass. BITS differs from canonical homologous recombination in that the RING, SAP, and UBZ domains of RAD18 were found to be indispensable, while for canonical homologous recombination both the E3 ligase activity-connected RING and the SAP (DNA-binding) domains are dispensable (Huang et al., 2009).