Ubiquitin and SUMO pathways in DNA replication and replication-coupled repair

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

We have previously reported that p53 decelerates nascent DNA elongation in complex with the translesion synthesis (TLS) polymerase ι (POLι) which triggers a homology-directed DNA damage tolerance (DDT) pathway to bypass obstacles during DNA replication. Here, we demonstrate that this DDT pathway relies on multiple p53 activities, which can be disrupted by TP53 mutations including those frequently found in cancer tissues. We show that the p53-mediated DDT pathway depends on its oligomerization domain (OD), while its regulatory C-terminus is not involved. Mutation of residues S315 and D48/D49, which abrogate p53 interactions with the DNA repair and replication proteins topoisomerase I and RPA, respectively, and residues L22/W23, which disrupt formation of p53-POLι complexes, all prevent this DDT pathway. Our results demonstrate that the p53-mediated DDT requires the formation of a DNA binding-proficient p53 tetramer, recruitment of such tetramer to RPA-coated forks and p53 complex formation with POLι. Importantly, our mutational analysis demonstrates that transcriptional transactivation is dispensable for the POLι-mediated DDT pathway, which we show protects against DNA replication damage from endogenous and exogenous sources.

Phosphorylation of human INO80 is involved in DNA damage tolerance

Perturbations in folate-mediated one-carbon metabolism increase rates of uracil misincorporation into DNA during replication, impair cellular methylation reactions, and increase risk for neural tube defects and cancer. One-carbon metabolism is compromised by folate deficiency and common genetic polymorphisms. In this study, the mechanism for the preferential partitioning of cytoplasmic serine hydroxymethyltransferase (cSHMT)-derived methylenetetrahydrofolate to de novo thymidylate biosynthesis was investigated. The cSHMT enzyme was shown to interact with UBC9 and was a substrate for UBC9-catalyzed small ubiquitin-like modifier (SUMO) modification in vitro. SUMOylated cSHMT was detected in extracts from S phase MCF-7 cells, and cSHMT was shown to localize to the nucleus and nuclear periphery during the S and G(2)/M phases of the cell cycle. A common single nucleotide polymorphism (L474F-cSHMT) impaired the UBC9-cSHMT interaction and inhibited cSHMT SUMOylation in vitro. The three folate-dependent enzymes that constitute the de novo thymidylate biosynthesis pathway, cSHMT, thymidylate synthase, and dihydrofolate reductase, all contain SUMO modification consensus sequences. Compartmentation of the folate-dependent de novo thymidylate biosynthesis pathway in the nucleus accounts for the preferential partitioning of cSHMT-derived folate-activated one-carbon units into thymidylate biosynthesis; the efficiency of nuclear folate metabolism is likely to be modified by the cSHMT L474F polymorphism.

The recently discovered p53-dependent DNA damage tolerance (DDT) pathway relies on its biochemical activities in DNA-binding, oligomerization, as well as complex formation with the translesion synthesis (TLS) polymerase iota (POLι). These p53-POLι complexes slow down nascent DNA synthesis for safe, homology-directed bypass of DNA replication barriers. In this study, we demonstrate that the alternative p53-isoforms p53β, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α differentially affect this p53-POLι-dependent DDT pathway originally described for canonical p53α. We show that the C-terminal isoforms p53β and p53γ, comprising a truncated oligomerization domain (OD), bind PCNA. Conversely, N-terminally truncated isoforms have a reduced capacity to engage in this interaction. Regardless of the specific loss of biochemical activities required for this DDT pathway, all alternative isoforms were impaired in promoting POLι recruitment to PCNA in the chromatin and in decelerating DNA replication under conditions of enforced replication stress after Mitomycin C (MMC) treatment. Consistent with this, all alternative p53-isoforms no longer stimulated recombination, i.e., bypass of endogenous replication barriers. Different from the other isoforms, Δ133p53α and Δ160p53α caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53α exacerbated the DDT pathway defects, unveiling impaired POLι recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53α was particularly pronounced in cells co-expressing Δ133p53α or Δ160p53α. Notably, this effect became evident after the expression of the isoforms in tumor cells, as well as after the knockdown of endogenous isoforms in human hematopoietic stem and progenitor cells. In summary, mimicking the situation found to be associated with many cancer types and stem cells, i.e., co-expression of alternative p53-isoforms with p53α, carved out interference with p53α functions in the p53-POLι-dependent DDT pathway.

Sumoylation of PCNA: Wrestling with recombination at stalled replication forks

ATP-dependent chromatin remodeling complexes have been shown to participate in DNA replication in addition to transcription and DNA repair. However, the mechanisms of their involvement in DNA replication remain unclear. Here, we reveal a specific function of the yeast INO80 chromatin remodeling complex in the DNA damage tolerance pathways. Whereas INO80 is necessary for the resumption of replication at forks stalled by methyl methane sulfonate (MMS), it is not required for replication fork collapse after treatment with hydroxyurea (HU). Mechanistically, INO80 regulates DNA damage tolerance during replication through modulation of PCNA (proliferating cell nuclear antigen) ubiquitination and Rad51-mediated processing of recombination intermediates at impeded replication forks. Our findings establish a mechanistic link between INO80 and DNA damage tolerance pathways, indicating that chromatin remodeling is important for accurate DNA replication.

DNA polymerases sometimes stall during DNA replication at sites where DNA is damaged, or upon encounter with proteins or secondary structures of DNA. When that happens, the polymerase clamp PCNA can become modified with a single ubiquitin moiety at lysine 164, opening DNA Damage Tolerance (DDT) mechanisms that either repair or bypass the lesions. An alternative repair mechanism is the salvage recombination (SR) pathway, which copies information from the sister chromatid. SUMOylation of PCNA at the same lysine, or at lysine 127, can recruit the Srs2 helicase, which negatively controls SR. Recently, we have dissected the relationship between SR and the DDT pathways, and showed that overexpression of either the PCNA unloader Elg1, or the Rad52 homologous recombination protein, can bypass the repression by Srs2. Our results shed light on the interactions between different DNA damage repair/bypass proteins, and underscore the importance of PCNA modifications in organizing the complex task of dealing with DNA damage during replication of the genetic material.

DNA lesions arising from both exogenous and endogenous sources occur frequently in DNA. During DNA replication, the presence of unrepaired DNA damage in the template can arrest replication fork progression, leading to fork collapse, double-strand break formation, and to genome instability. To facilitate completion of replication and prevent the generation of strand breaks, DNA damage tolerance (DDT) pathways play a key role in allowing replication to proceed in the presence of lesions in the template. The two main DDT pathways are translesion synthesis (TLS), which involves the recruitment of specialized TLS polymerases to the site of replication arrest to bypass lesions, and homology-directed damage tolerance, which includes the template switching and fork reversal pathways. With some exceptions, lesion bypass by TLS polymerases is a source of mutagenesis, potentially contributing to the development of cancer. The capacity of TLS polymerases to bypass replication-blocking lesions induced by anti-cancer drugs such as cisplatin can also contribute to tumor chemoresistance. On the other hand, during homology-directed DDT the nascent sister strand is transiently utilised as a template for replication, allowing for error-free lesion bypass. Given the role of DNA damage tolerance pathways in replication, mutagenesis and chemoresistance, a more complete understanding of these pathways can provide avenues for therapeutic exploitation. A number of small molecule inhibitors of TLS polymerase activity have been identified that show synergy with conventional chemotherapeutic agents in killing cancer cells. In this review, we will summarize the major DDT pathways, explore the relationship between damage tolerance and carcinogenesis, and discuss the potential of targeting TLS polymerases as a therapeutic approach.

DNA lesion bypass is mediated by DNA damage tolerance (DDT) pathways and homologous recombination (HR). The DDT pathways, which involve translesion synthesis and template switching (TS), are activated by the ubiquitylation (ub) of PCNA through components of the RAD6-RAD18 pathway, whereas the HR pathway is independent of RAD18 However, it is unclear how these processes are coordinated within the context of chromatin. Here we show that Bre1, an ubiquitin ligase specific for histone H2B, is recruited to chromatin in a manner coupled to replication of damaged DNA. In the absence of Bre1 or H2Bub, cells exhibit accumulation of unrepaired DNA lesions. Consequently, the damaged forks become unstable and resistant to repair. We provide physical, genetic, and cytological evidence that H2Bub contributes toward both Rad18-dependent TS and replication fork repair by HR. Using an inducible system of DNA damage bypass, we further show that H2Bub is required for the regulation of DDT after genome duplication. We propose that Bre1-H2Bub facilitates fork recovery and gap-filling repair by controlling chromatin dynamics in response to replicative DNA damage.

HUWE1 is a multi‐faceted E3 ubiquitin ligase of the HECT family with many confirmed substrates, but mechanistic understanding of its functional roles in signaling pathways remains limited. In this issue of EMBO Reports, Choe et al demonstrate a novel function for HUWE1 in promoting DNA damage tolerance mechanisms to bypass DNA lesions during replication stress, thereby preserving genome stability. The authors connect this role for HUWE1 with its function in maintaining H2AX monoubiquitination levels for efficient signaling at stalled replication forks . Thus, this work highlights HUWE1 as a novel player in the replication stress response and prompts further investigation of its regulation during replication and other cellular processes.

The Intra-S Phase Checkpoint Targets Dna2 to Prevent Stalled Replication Forks from Reversing

DNA repair is essential to maintain genome integrity. In addition to various DNA repair pathways dealing with specific types of DNA lesions, DNA damage tolerance (DDT) promotes the bypass of DNA replication blocks encountered by the replication fork to prevent cell death. Budding yeast Rad5 plays an essential role in the DDT pathway and its structure indicates that Rad5 recognizes damaged DNA or stalled replication forks, suggesting that Rad5 plays an important role in the DDT pathway choice. It has been reported that Rad5 forms subnuclear foci in the presence of methyl methanesulfonate (MMS) during the S phase. By analyzing the formation of Rad5 foci after MMS treatment, we showed that some specific DNA structures rather than mono-ubiquitination of proliferating cell nuclear antigen are required for the recruitment of Rad5 to the damaged site. Moreover, inactivation of the base excision repair (BER) pathway greatly decreased the Rad5 focus formation, suggesting that Rad5 recognizes specific DNA structures generated by BER. We also identified a negative role of overexpressed translesion synthesis polymerase Polη in the formation of Rad5 foci. Based on these data, we propose a modified DDT pathway model in which Rad5 plays a role in activating the DDT pathway.

Chk1 is a conserved kinase that imposes cell cycle delays in response to impediments to DNA replication. Recent experiments have further defined effects of Chk1 on the activity of mammalian origins of DNA replication and progression of replication forks. Moreover, Chk1 now appears to help defend genomic integrity through effects on several other pathways, including Fanconi anemia proteins, the mitotic spindle, and transcription of cell cycle-related genes. These findings can account for the requirement for Chk1 in normal proliferating cells of the early embryo and suggest the potential for diverse effects of Chk1 inhibition in cancer therapy.

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.