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Please note this is a comparison between Version 1 by Gemma Belli Martinez and Version 2 by Lindsay Dong.
The sliding clamp proliferating cell nuclear antigen (PCNA) is a multifunctional homotrimer mainly linked to DNA replication. During this process, cells must ensure an accurate and complete genome replication when constantly challenged by the presence of DNA lesions. Post-translational modifications of PCNA play a crucial role in channeling DNA damage tolerance (DDT) and repair mechanisms to bypass unrepaired lesions and promote optimal fork replication restart. PCNA ubiquitination processes trigger the following two main DDT sub-pathways: Rad6/Rad18-dependent PCNA monoubiquitination and Ubc13-Mms2/Rad5-mediated PCNA polyubiquitination, promoting error-prone translation synthesis (TLS) or error-free template switch (TS) pathways, respectively. However, the fork protection mechanism leading to TS during fork reversal is still poorly understood. In contrast, PCNA sumoylation impedes the homologous recombination (HR)-mediated salvage recombination (SR) repair pathway.
- PCNA
- DNA damage tolerance
- DNA replication stress
- fungal genome stability
1. Proliferating Cell Nuclear Antigen (PCNA)
The sliding clamp proliferating cell nuclear antigen (PCNA), encoded by POL30 in S. cerevisiae [1], is central to DNA replication and repair [2][3][2,3], and other fundamental processes of DNA metabolism, such as chromatin assembly [4][5][6][4,5,6], sister chromatid cohesion establishment [7][8][9][7,8,9], lagging strand maturation [10][11][10,11], epigenetic conversions and inheritance [12][13][14][12,13,14], gene expression [15][16][15,16] and DNA damage checkpoint pathway activation [17]. Initially, PCNA was discovered as a biological marker for systemic lupus erythematosus disease [18]. In the early eighties, PCNA was identified as a cyclin [19][20][19,20] due to its role in proliferation and correlative expression with the cell cycle. Subsequently, it was characterized as a processivity factor for replicative DNA polymerase (Pol) δ [21][22][23][21,22,23], Polα and β [24], and Polε [25][26][25,26]. Therefore, PCNA was related to DNA replication [27] and it was given essential roles on the replication fork (RF), including leading- and lagging-strand synthesis coordination in combination with replication factor C (RFC) [24], and the synthesis of the leading strand during the elongation stage [28]. PCNA was soon associated with nucleotide excision repair [29][30][29,30], together with the role of DNA Polδ in UV-induced DNA damage [31][32][31,32]. Concomitantly, the function PCNA in DNA damage repair was elucidated [33][34][35][33,34,35]. Indeed, PCNA has become a major scaffold protein for DNA damage response by participating in multiple DNA repair pathways, including DNA mismatch repair [36][37][38][36,37,38], base excision repair (BER) [39][40][41][42][39,40,41,42], double-strand break repair [43][44][45][43,44,45], translesion synthesis (TLS) [46][47][48][49][50][46,47,48,49,50], the resolution of deleterious interactions between replication and transcription machinery [51], and in the post-translational regulation of DNA damage tolerance (DDT) through a selection of various sub-pathways of the post-replication repair pathway [52].
2. PCNA Structural Features
PCNA belongs to the conserved family of DNA sliding clamps (β clamps) [53][54][55][56][53,54,55,56]. It forms a homotrimer in a closed head-to-tail ring structure around DNA. Each PCNA molecule has two globular domains linked by the inter-domain connecting loop (IDCL) [56]. The PCNA homotrimer is disposed in a pseudo-six-fold symmetry structure with an external layer of six β-sheets and an internal layer of twelve α-helices. The negatively charged outer surface potentially inhibits unspecific protein interactions, while the positively charged inner surface of the clamp structure faces the DNA duplex [53][54][55][53,54,55]. This interaction occurs through five basic lysine residues sliding on successive phosphates of one DNA strand by a spiral movement [57][58][57,58]. Since DNA is a helical molecule, the sliding of the PCNA homotrimer is tilted 30° and moves forward to backward every one-half turn of DNA in a process defined as “cogwheeling” [58]. The clamp tilt at the 3′ end of the DNA molecule may specify PCNA binding partners [59]. Another proposed mechanism for sliding is the “switch” from five of the twelve α-helices tracked by the DNA, which consequently changes the relative tilt to the 3′ terminus [57]. There exists a dynamic association/dissociation process, in which RFC loads PCNA onto RFs for its interaction with replicative Polδ [60]. In S. cerevisiae, PCNA only interacts with Pol3 (the catalytic subunit of Polδ) providing enough room between the inner walls of PCNA and the DNA to hold water molecules, which may facilitate sliding [61]. Once the polymerase dissociates, PCNA diffuses away from the primer terminus. Later, it re-associates with the polymerase to finish replication. This back-and-forward clamp motion has been suggested as the possible Polδ proofreading activity mechanism [58] or even the switch allowing translesion DNA polymerase to bypass DNA lesions [59]. Additionally, post-translational modifications also occur on the surface of the PCNA clamp to regulate DNA sliding [62][63][62,63] and in response to DNA damage [64]. PCNA functions as a platform for a large amount of DNA replication and editing enzymes [65][66][65,66]. Many of the PCNA interactors exhibit a short linear motif (SLiM) [67]. The PCNA binding motif is known as the PCNA-interacting peptide (PIP) box [68], through which many PCNA-interacting proteins are recruited to function in DNA replication [69]. Its extended PIP degron version targets PCNA for degradation [70]. A second important interaction motif is the AlkB homologue 2 PCNA interacting motif (APIM) [71][72][73][71,72,73], which mediates the interaction with genotoxic responsive proteins [69]. Both motifs are topologically similar and are localized in the hydrophobic pocket formed in the IDCL region of the β-sheets external surface of the ring [74][75][74,75]. Moreover, another PCNA interaction motif named the KA box has been described [76][77][76,77]. PCNA homotrimer may interact with three different partners synchronically through the three IDCL regions, facilitating spatio-temporal coordination for a multiplicity of purposes. Besides, the PIP box may overlap with the Rev1-interacting region (RIR)- and the Mlh-1 interacting protein (MIP) motifs [78], both of which include proteins related to DNA repair, enhancing the number of interactions with genome maintenance interactors (see details in the text). Interestingly, several PCNA binding partners are considered intrinsically disordered proteins (IDPs) or hold intrinsically disordered regions (IDRs) [79][80][79,80]. IDPs are unstructured in physiological conditions and fold to stable structures when binding their functional targets [81]. Rather abundant in eukaryotes, IDPs are relevant members of cellular signaling and become highly regulated at the post-translational level [82]. Therefore, the existence of three hydrophobic binding pockets in PCNA clamps, the redundancy of SLiM sequences, and the numerous IDPs interactors increase the complexity of the regulation of PCNA-mediated processes.3. Post-Translational Modifications of PCNA and the DDT Pathways
Endogenous processes, as well as exogenous DNA damaging agents, such as UV-irradiation and alkylating agents, may lead to the formation of bulky lesions that eventually stall RFs. Organisms have developed a collection of mechanisms to ensure the progression of DNA replication in the presence of DNA-disturbing events, the DDT pathways. The post-translational modification of PCNA triggers DNA damage response, mainly by determining the interacting proteins to be recruited. Three main DDT pathways can be involved, which are as follows: the translesion synthesis (TLS), template switch (TS) and salvage recombination (SR) pathways (Figure 12).
Figure 12. PCNA post-translational modifications regulate DDT pathways. When fork stalling persists, cells activate DDT mechanisms through post-translational modifications on PCNA. Monoubiquitination of PCNA at K164 by Rad6-Rad18 promotes the switch from Polδ to translesion synthesis (TLS) polymerases for error-prone TLS damage bypass. K63 extended polyubiquitination of PCNA on K164 by Mms2/Ubc13-Rad5 for error-free damage bypass mediates template switching (TS). This modification might be also implied in fork protection by fork reversal. Unloading of SUMO-PCNA bound to Srs2 (sumoylated by Ubc9-Siz1) provides the salvage recombination pathway (SR) alternatively to TS or TLS, either at stalled replication forks (RFs) or, as shown, at gaps left behind RFs after re-initiation. Cell cycle stages where DDT processes predominantly take place in yeast are indicated. Blocking DNA lesions are depicted as a red star.
3.1. Translesion Synthesis (TLS)-Mediated DDT Error-Prone Pathway
Bulky lesions may prevent DNA synthesis by classical replicative DNA polymerases, consequently leading to stalled RFs. In this scenario, genome instability and chromosome rearrangements may compromise cell viability. To avoid prolonged uncoupling, cells trigger a DDT mechanism termed TLS that allows replication to proceed through damaged DNA or stalled RFs. However, replicative DNA polymerases are not efficient at incorporating nucleotides when the opposite DNA template is damaged. Instead, TLS is executed by specialized TLS polymerases, which are less stringent to DNA damage and are able to synthesize across DNA lesions, with no associated proofreading activity. The decrease in fidelity may trigger the accumulation of mutations. In fact, TLS represents the major accumulation of mutations by an error-prone bypass pathway in eukaryotes [83][84][85][86][87][88][89][90][91][92][118,119,120,121,122,123,124,125,126,127].