The DHX9 helicase interacts with human DNA polymerase δ4 and stimulates its activity in D-loop extension synthesis.

The extension of the invading strand within a displacement loop (D-loop) is a key step in homology directed repair (HDR) of doubled stranded DNA breaks. The primary goal of these studies was to test the hypotheses that 1) D-loop extension by human DNA polymerase δ4 (Pol δ4) is facilitated by DHX9, a 3' to 5' motor helicase, which acts to unwind the leading edge of the D-loop, and 2) the recruitment of DHX9 is mediated by direct protein-protein interactions between DHX9 and Pol δ4 and/or PCNA. DNA synthesis by Pol δ4 was analyzed in a reconstitution assay by the extension of a 93mer oligonucleotide inserted into a plasmid to form a D-loop. Product formation by Pol δ4 was monitored by incorporation of [α-32P]dNTPs into the 93mer primer followed by denaturing gel electrophoresis. The results showed that DHX9 strongly stimulated Pol δ4 mediated D-loop extension. Direct interactions of DHX9 with PCNA, the p125 and the p12 subunits of Pol δ4 were demonstrated by pull-down assays with purified proteins. These data support the hypothesis that DHX9 helicase is recruited by Pol δ4/PCNA to facilitate D-loop synthesis in HDR, and is a participant in cellular HDR. The involvement of DHX9 in HDR represents an important addition to its multiple cellular roles. Such helicase-polymerase interactions may represent an important aspect of the mechanisms involved in D-loop primer extension synthesis in HDR.

Special Issue "DNA Replication/Repair, and the DNA Damage Response in Human Disease".

Mutations of numerous genes involved in DNA replication, DNA repair, and DNA damage response (DDR) pathways lead to a variety of human diseases, including aging and cancer [...].

POLDIP3: At the Crossroad of RNA and DNA Metabolism.

POLDIP3 was initially identified as a DNA polymerase delta (Pol δ) interacting protein almost twenty years ago. Intriguingly, it also interacts with proteins involved in a variety of RNA related biological processes, such as transcription, pre-mRNA splicing, mRNA export, and translation. Studies in recent years revealed that POLDIP3 also plays critical roles in disassembling genome wide R-loop formation and activating the DNA damage checkpoint in vivo. Here, we review the functions of POLDIP3 in various RNA and DNA related cellular processes. We then propose a unified model to illustrate how POLDIP3 plays such a versatile role at the crossroad of the RNA and DNA metabolism.

DNA polymerase delta interacting protein 3 facilitates the activation and maintenance of DNA damage checkpoint in response to replication stress.

BACKGROUND: Replication stress response is crucial for the maintenance of a stable genome. POLDIP3 (DNA polymerase delta interacting protein 3) was initially identified as one of the DNA polymerase δ (Pol δ) interacting proteins almost 20 years ago. Using a variety of in vitro biochemical assays, we previously established that POLDIP3 is a key regulator of the enzymatic activity of Pol δ. However, the in vivo function of POLDIP3 in DNA replication and DNA damage response has been elusive. METHODS: We first generated POLDIP3 knockout (KO) cells using the CRISPR/Cas9 technology. We then investigated its biological functions in vivo using a variety of biochemical and cell biology assays. RESULTS: We showed that although the POLDIP3-KO cells manifest no pronounced defect in global DNA synthesis under nonstress conditions, they are sensitive to a variety of replication fork blockers. Intriguingly, we found that POLDIP3 plays a crucial role in the activation and maintenance of the DNA damage checkpoint in response to exogenous as well as endogenous replication stress. CONCLUSION: Our results indicate that when the DNA replication fork is blocked, POLDIP3 can be recruited to the stalled replication fork and functions to bridge the early DNA damage checkpoint response and the later replication fork repair/restart.

A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo.

In many repeat diseases, such as Huntington's disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSß. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats.

Two forms of human DNA polymerase δ: Who does what and why?

DNA polymerase δ (Pol δ) plays a central role in lagging strand DNA synthesis in eukaryotic cells, as well as an important role in DNA repair processes. Human Pol δ4 is a heterotetramer of four subunits, the smallest of which is p12. Pol δ3 is a trimeric form that is generated in vivo by the degradation of the p12 subunit in response to DNA damage, and during entry into S-phase. The biochemical properties of the two forms of Pol δ, as well as the changes in their distribution during the cell cycle, are reviewed from the perspective of understanding their respective cellular functions. Biochemical and cellular studies support a role for Pol δ3 in gap filling during DNA repair, and in Okazaki fragment synthesis during DNA replication. Recent studies of cells in which p12 expression is ablated, and are therefore null for Pol δ4, show that Pol δ4 is not required for cell viability. These cells have a defect in homologous recombination, revealing a specific role for Pol δ4 that cannot be performed by Pol δ3. Pol δ4 activity is required for D-loop displacement synthesis in HR. The reasons why Pol δ4 but not Pol δ3 can perform this function are discussed, as well as the question of whether helicase action is needed for efficient D-loop displacement synthesis. Pol δ4 is largely present in the G1 and G2/M phases of the cell cycle and is low in S phase. This is discussed in relation to the availability of Pol δ4 as an additional layer of regulation for HR activity during cell cycle progression.

Loss of the p12 subunit of DNA polymerase delta leads to a defect in HR and sensitization to PARP inhibitors.

Human DNA polymerase δ is normally present in unstressed, non-dividing cells as a heterotetramer (Pol δ4). Its smallest subunit, p12, is transiently degraded in response to UV damage, as well as during the entry into S-phase, resulting in the conversion of Pol δ4 to a trimer (Pol δ3). In order to further understand the specific cellular roles of these two forms of Pol δ, the gene (POLD4) encoding p12 was disrupted by CRISPR/Cas9 to produce p12 knockout (p12KO) cells. Thus, Pol δ4 is absent in p12KO cells, leaving Pol δ3 as the sole source of Pol δ activity. GFP reporter assays revealed that the p12KO cells exhibited a defect in homologous recombination (HR) repair, indicating that Pol δ4, but not Pol δ3, is required for HR. Expression of Flag-tagged p12 in p12KO cells to restore Pol δ4 alleviated the HR defect. These results establish a specific requirement for Pol δ4 in HR repair. This leads to the prediction that p12KO cells should be more sensitive to chemotherapeutic agents, and should exhibit synthetic lethal killing by PARP inhibitors. These predictions were confirmed by clonogenic cell survival assays of p12KO cells treated with cisplatin and mitomycin C, and with the PARP inhibitors Olaparib, Talazoparib, Rucaparib, and Niraparib. The sensitivity to PARP inhibitors in H1299-p12KO cells was alleviated by expression of Flag-p12. These findings have clinical significance, as the expression levels of p12 could be a predictive biomarker of tumor response to PARP inhibitors. In addition, small cell lung cancers (SCLC) are known to exhibit a defect in p12 expression. Analysis of several SCLC cell lines showed that they exhibit hypersensitivity to PARP inhibitors, providing evidence that loss of p12 expression could represent a novel molecular basis for HR deficiency.

Discovery of a novel DNA polymerase inhibitor and characterization of its antiproliferative properties.

Chromosomal duplication is targeted by various chemotherapeutic agents for the treatment of cancer. However, there is no specific inhibitor of DNA polymerases that is viable for cancer management. Through structure-based in silico screening of the ZINC database, we identified a specific inhibitor of DNA polymerase δ. The discovered inhibitor, Zelpolib, is projected to bind to the active site of Pol δ when it is actively engaged in DNA replication through interactions with DNA template and primer. Zelpolib shows robust inhibition of Pol δ activity in reconstituted DNA replication assays. Under cellular conditions, Zelpolib is taken up readily by cancer cells and inhibits DNA replication in assays to assess global DNA synthesis or single-molecule bases by DNA fiber fluorography. In addition, we show that Zelpolib displays superior antiproliferative properties to methotrexate, 5-flourouracil, and cisplatin in triple-negative breast cancer cell line, pancreatic cancer cell line and platinum-resistant pancreatic cancer cell line. Pol δ is not only involved in DNA replication, it is also a key component in many DNA repair pathways. Pol δ is the key enzyme responsible for D-loop extension during homologous recombination. Indeed, Zelpolib shows robust inhibition of homologous recombination repair of DNA double-strand breaks and induces "BRCAness" in HR-proficient cancer cells and enhances their sensitivity to PARP inhibitors.

Phosphorylation Alters the Properties of Pol η: Implications for Translesion Synthesis.

There are significant ambiguities regarding how DNA polymerase η is recruited to DNA lesion sites in stressed cells while avoiding normal replication forks in non-stressed cells. Even less is known about the mechanisms responsible for Pol η-induced mutations in cancer genomes. We show that there are two safeguards to prevent Pol η from adventitious participation in normal DNA replication. These include sequestration by a partner protein and low basal activity. Upon cellular UV irradiation, phosphorylation enables Pol η to be released from sequestration by PDIP38 and activates its polymerase function through increased affinity toward monoubiquitinated proliferating cell nuclear antigen (Ub-PCNA). Moreover, the high-affinity binding of phosphorylated Pol η to Ub-PCNA limits its subsequent displacement by Pol δ. Consequently, activated Pol η replicates DNA beyond the lesion site and potentially introduces clusters of mutations due to its low fidelity. This mechanism could account for the prevalence of Pol η signatures in cancer genome.

Regulation and Modulation of Human DNA Polymerase δ Activity and Function.

This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on the mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, polymerase delta interaction protein 46 (PDIP46) and polymerase delta interaction protein 38 (PDIP38), both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the spliceosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, mouse double minute 2 homolog (Mdm2) alternative splicing and the regulation of the NADPH oxidase 4 (Nox4).

DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis.

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA.

PDIP46 (DNA polymerase δ interacting protein 46) is an activating factor for human DNA polymerase δ.

PDIP46 (SKAR, POLDIP3) was discovered through its interaction with the p50 subunit of human DNA polymerase δ (Pol δ). Its functions in DNA replication are unknown. PDIP46 associates with Pol δ in cell extracts both by immunochemical and protein separation methods, as well as by ChIP analyses. PDIP46 also interacts with PCNA via multiple copies of a novel PCNA binding motif, the APIMs (AlkB homologue-2 PCNA-Interacting Motif). Sites for both p50 and PCNA binding were mapped to the N-terminal region containing the APIMs. Functional assays for the effects of PDIP46 on Pol δ activity on singly primed ssM13 DNA templates revealed that it is a novel and potent activator of Pol δ. The effects of PDIP46 on Pol δ in primer extension, strand displacement and synthesis through simple hairpin structures reveal a mechanism where PDIP46 facilitates Pol δ4 synthesis through regions of secondary structure on complex templates. In addition, evidence was obtained that PDIP46 is also capable of exerting its effects by a direct interaction with Pol δ, independent of PCNA. Mutation of the Pol δ and PCNA binding region resulted in a loss of PDIP46 functions. These studies support the view that PDIP46 is a novel accessory protein for Pol δ that is involved in cellular DNA replication. This raises the possibility that altered expression of PDIP46 or its mutation may affect Pol δ functions in vivo, and thereby be a nexus for altered genomic stability.

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

Genomically Incorporated 5-Fluorouracil that Escapes UNG-Initiated Base Excision Repair Blocks DNA Replication and Activates Homologous Recombination.

5-Fluorouracil (5-FU) and its metabolite 5-fluorodeoxyuridine (FdUrd, floxuridine) are chemotherapy agents that are converted to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP). FdUMP inhibits thymidylate synthase and causes the accumulation of uracil in the genome, whereas FdUTP is incorporated by DNA polymerases as 5-FU in the genome; however, it remains unclear how either genomically incorporated U or 5-FU contributes to killing. We show that depletion of the uracil DNA glycosylase (UNG) sensitizes tumor cells to FdUrd. Furthermore, we show that UNG depletion does not sensitize cells to the thymidylate synthase inhibitor (raltitrexed), which induces uracil but not 5-FU accumulation, thus indicating that genomically incorporated 5-FU plays a major role in the antineoplastic effects of FdUrd. We also show that 5-FU metabolites do not block the first round of DNA synthesis but instead arrest cells at the G1/S border when cells again attempt replication and activate homologous recombination (HR). This arrest is not due to 5-FU lesions blocking DNA polymerase δ but instead depends, in part, on the thymine DNA glycosylase. Consistent with the activation of HR repair, disruption of HR sensitized cells to FdUrd, especially when UNG was disabled. These results show that 5-FU lesions that escape UNG repair activate HR, which promotes cell survival.

Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry.

During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

The tail that wags the dog: p12, the smallest subunit of DNA polymerase δ, is degraded by ubiquitin ligases in response to DNA damage and during cell cycle progression.

DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication. Human Pol δ is a heterotetramer whose p12 subunit is degraded in response to DNA damage, leading to the in vivo conversion of Pol δ4 to Pol δ3. Two E3 ubiquitin ligases, RNF8 and CRL4(Cdt2), participate in the DNA damage-induced degradation of p12. We discuss how these E3 ligases integrate the formation of Pol δ3 and ubiquitinated PCNA for DNA repair processes. CRL4(Cdt2) partially degrades p12 during normal cell cycle progression, thereby generating Pol δ3 during S phase. This novel finding extends the current view of the role of Pol δ3 in DNA repair and leads to the hypothesis that it participates in DNA replication. The coordinated regulation of licensing factors and Pol δ3 by CRL4(Cdt2) now opens new avenues for control of DNA replication. A parallel study of Pol δ4 and Pol δ3 in Okazaki fragment processing provides evidence for a role of Pol δ3 in DNA replication. We discuss several new perspectives of the role of the 2 forms of Pol δ in DNA replication and repair, as well the significance of the integration of p12 regulation in DNA repair and cell cycle progression.

DNA polymerase δ stalls on telomeric lagging strand templates independently from G-quadruplex formation.

Previous evidence indicates that telomeres resemble common fragile sites and present a challenge for DNA replication. The precise impediments to replication fork progression at telomeric TTAGGG repeats are unknown, but are proposed to include G-quadruplexes (G4) on the G-rich strand. Here we examined DNA synthesis and progression by the replicative DNA polymerase δ/proliferating cell nuclear antigen/replication factor C complex on telomeric templates that mimic the leading C-rich and lagging G-rich strands. Increased polymerase stalling occurred on the G-rich template, compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting Li(+) for K(+) as the cation, or by using templates with 7-deaza-G residues, did not alleviate Pol δ pause sites within the G residues. Furthermore, we provide evidence that G4 folding is less stable on single-stranded circular TTAGGG templates where ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on the circular templates with the G4 ligand BRACO-19 inhibited Pol δ progression into the G-rich repeats. Similar results were obtained for yeast and human Pol δ complexes. Our data indicate that G4 formation is not required for polymerase stalling on telomeric lagging strands and suggest that an alternative mechanism, in addition to stable G4s, contributes to replication stalling at telomeres.

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ.

Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5'-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1-10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells.

A novel function of CRL4(Cdt2): regulation of the subunit structure of DNA polymerase δ in response to DNA damage and during the S phase.

DNA polymerase δ (Pol δ4) is a heterotetrameric enzyme, whose p12 subunit is degraded in response to DNA damage, leaving behind a trimer (Pol δ3) with altered enzymatic characteristics that participate in gap filling during DNA repair. We demonstrate that CRL4(Cdt2), a key regulator of cell cycle progression that targets replication licensing factors, also targets the p12 subunit of Pol δ4 in response to DNA damage and on entry into S phase. Evidence for the involvement of CRL4(Cdt2) included demonstration that p12 possesses a proliferating cell nuclear antigen-interacting protein-degron (PIP-degron) and that knockdown of the components of the CRL4(Cdt2) complex inhibited the degradation of p12 in response to DNA damage. Analysis of p12 levels in synchronized cell populations showed that p12 is partially degraded in S phase and that this is affected by knockdowns of CUL4A or CUL4B. Laser scanning cytometry of overexpressed wild type p12 and a mutant resistant to degradation showed that the reduction in p12 levels during S phase was prevented by mutation of p12. Thus, CRL4(Cdt2) also regulates the subunit composition of Pol δ during the cell cycle. These studies reveal a novel function of CRL4(Cdt2), i.e. the direct regulation of DNA polymerase δ, adding to its known functions in the regulation of the licensing of replication origins and expanding the scope of its overall control of DNA replication. The formation of Pol δ3 in S phase as a normal aspect of cell cycle progression leads to the novel implications that it is involved in DNA replication as well as DNA repair.

PDIP38 is translocated to the spliceosomes/nuclear speckles in response to UV-induced DNA damage and is required for UV-induced alternative splicing of MDM2.

PDIP38 (polymerase delta interacting protein 38) was originally discovered as a protein that interacts with DNA polymerase δ and PCNA. PDIP38 is present in multiple intracellular locations and is a multifunctional protein that has been implicated in several diverse cellular functions. We investigated the nuclear localization of PDIP38 in order to gain insights to its response to UV damage. PDIP38 was found to form distinct nuclear foci in response to UV irradiation in several cell lines, including HeLa S3 and A549 cells. However, these foci were not those associated with UV repair foci. Using various markers for different nuclear subcompartments, the UV-induced PDIP38 foci were identified as spliceosomes/nuclear speckles, the storage and assembly sites for mRNA splicing factors. To assess the role of PDIP38 in the regulation of splicing events, the effects of PDIP38 depletion on the UV-induced alternate splicing of MDM2 transcripts were examined by nested RT-PCR. Alternatively spliced MDM2 products were induced by UV treatment but were greatly reduced in cells expressing shRNA targeting PDIP38. These findings indicate that upon UV-induced DNA damage, PDIP38 is translocated to spliceosomes and contributes to the UV-induced alternative splicing of MDM2 transcripts. Similar results were obtained when cells were subjected to transcriptional stresses with actinomycin D or α-amanitin. Taken together, these studies show that PDIP38 is a protein regulated in a dynamic manner in response to genotoxic stress, as evidenced by its translocation to the spliceosomes. Moreover, PDIP38 is required for the induction of the alternative splicing of MDM2 in response to UV irradiation.