APIM-Mediated REV3L–PCNA Interaction Important for Error Free TLS Over UV-Induced DNA Lesions in Human Cells

Abstract

Proliferating cell nuclear antigen (PCNA) is essential for the organization of DNA replication and the bypass of DNA lesions via translesion synthesis (TLS). TLS is mediated by specialized DNA polymerases, which all interact, directly or indirectly, with PCNA. How interactions between the TLS polymerases and PCNA affects TLS specificity and/or coordination is not fully understood. Here we show that the catalytic subunit of the essential mammalian TLS polymerase POLζ, REV3L, contains a functional AlkB homolog 2 PCNA interacting motif, APIM. APIM from REV3L fused to YFP, and full-length REV3L-YFP colocalizes with PCNA in replication foci. Colocalization of REV3L-YFP with PCNA is strongly reduced when an APIM-CFP construct is overexpressed. We also found that overexpression of full-length REV3L with mutated APIM leads to significantly altered mutation frequencies and mutation spectra, when compared to overexpression of full-length REV3L wild-type (WT) protein in multiple cell lines. Altogether, these data suggest that APIM is a functional PCNA-interacting motif in REV3L, and that the APIM-mediated PCNA interaction is important for the function and specificity of POLζ in TLS. Finally, a PCNA-targeting cell-penetrating peptide, containing APIM, reduced the mutation frequencies and changed the mutation spectra in several cell lines, suggesting that efficient TLS requires coordination mediated by interactions with PCNA.

Targeting the non-canonical roles of PCNA modifies and increases the response to targeted anti-cancer therapy

“…The ATX-101/AEE788 combination significantly reduced the percentage of viable 67NR cells compared to single treatments in vitro ( Figure 1A). We have previously shown that the biological effects of ATX-101 depends on its PCNA affinity, and that a peptide with reduced PCNA binding affinity (ATX-A) has no biological effect [17,18,21,22]. Also in this study, ATX-A had much lower effect on viability than ATX-101, and importantly did not enhance the growth inhibiting effect of AEE788 ( Figure 1A).…”

“…php), and several APIM-containing proteins are involved in the MAPK and PI3K/Akt pathways acting downstream of VEGFR and EGFR [20]. A cell-penetrating APIMcontaining peptide (ATX-101) can disrupt PCNA from interacting with APIM-containing proteins [17,21,22]. ATX-101 may therefore simultaneously impair several signaling pathways considered therapeutic targets in multiple cancers.…”

“…ATX-101 may therefore simultaneously impair several signaling pathways considered therapeutic targets in multiple cancers. ATX-101 has previously been shown to enhance the efficacy of different chemotherapeutics in in vivo cancer models [23][24][25], to inhibit mutagenesis by impairing DNA translesion synthesis (TLS) [22] and to modulate the PI3K/Akt and MAPK pathways [17]. Thus, ATX-101 could potentially both enhance, and prolong the efficacy of targeted therapies.…”

Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA)

“…To further investigate the importance of APIM in HLTF for colocalization with PCNA in replication foci, HLTF and HLTF F960A were overexpressed together with APIM-peptides and PCNA. The intensity of YFP-HLTF in PCNA foci was significantly reduced by co-expression of KFIVK-CFP (APIM in HLTF), and an even stronger reduction could be achieved by overexpression of RWLVK-CFP, an APIM-version with increased PCNA-affinity [36] ( Figure 1E,F, quantified in G). The intensity of YFP-HLTF F960A in PCNA foci was initially stronger than YFP-HLTF; however, after overexpression of APIM-peptides (KFIVK-CFP or RWLVK-CFP), foci intensity was reduced to the same or lower level as measured for YFP-HLTF ( Figure 1I,J, quantified in K).…”

“…Data suggest that cellular stress, e.g., such as replication stress, is important for increased APIM-PCNA affinity [17,24,26,27,[32][33][34]. Indeed, functional APIM is verified in three proteins directly involved in DDT, ZRANB3 [17], TFII-I [24,35], and the catalytic subunit of POLζ, REV3L [36], and in two proteins involved in the regulation of TS, FBH1 [28], and RAD51B [24]. Here we show that APIM in both HLTF and SHPRH interact with PCNA, and that the binding of HLTF and SHPRH to PCNA is important for their function in DDT after UV-induced DNA damage.…”