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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans.

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized.

DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis.

Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation-induced genome damage. We have recently observed that Pol η is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role.

Identification of RNF8 as a ubiquitin ligase involved in targeting the p12 subunit of DNA polymerase δ for degradation in response to DNA damage.

DNA polymerase δ consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) that are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation.

Regulation of human DNA polymerase delta in the cellular responses to DNA damage.

The p12 subunit of polymerase delta (Pol δ) is degraded in response to DNA damage induced by UV, alkylating agents, oxidative, and replication stresses. This leads to the conversion of the Pol δ4 holoenzyme to the heterotrimer, Pol δ3. We review studies that establish that Pol δ3 formation is an event that could have a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair.

Spatiotemporal recruitment of human DNA polymerase delta to sites of UV damage.

Human DNA polymerase δ (Pol δ) is involved in various DNA damage responses in addition to its central role in DNA replication. The Pol δ4 holoenzyme consists of four subunits, p125, p50, p68 and p12. It has been established that the p12 subunit is rapidly degraded in response to DNA damage by UV leading to the in vivo conversion of Pol δ4 to Pol δ3, a trimeric form lacking the p12 subunit.

Characterization of human DNA polymerase delta and its subassemblies reconstituted by expression in the MultiBac system.

Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit.

Structure of monoubiquitinated PCNA: implications for DNA polymerase switching and Okazaki fragment maturation.

Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as ub-PCNA orchestrates the recruitment and switching of translesion synthesis (TLS) polymerases with replication polymerases. This allows replication to proceed, leaving the DNA to be repaired subsequently. Defects in a TLS polymerase, Pol η, lead to a form of Xeroderma pigmentosum, a disease characterized by severe skin sensitivity to sunlight damage and an increased incidence of skin cancer.

Phosphorylation of the p68 subunit of Pol δ acts as a molecular switch to regulate its interaction with PCNA.

DNA polymerase delta (Pol δ) is a central enzyme for eukaryotic DNA replication and repair. Pol δ is a complex of four subunits p125, p68, p50, and p12. The functional properties of Pol δ are largely determined by its interaction with its DNA sliding clamp PCNA (proliferating cellular nuclear antigen).

P50, the small subunit of DNA polymerase delta, is required for mediation of the interaction of polymerase delta subassemblies with PCNA.

Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA.