The p12 Subunit Choreographs the Regulation and Functions of Two Forms of DNA Polymerase δ in Mammalian Cells

Abstract

There are two forms of DNA polymerase δ in human cells, Pol δ4 and Pol δ3, which differ based on their possession of the p12 subunit. The degradation of p12 has emerged as an important regulatory mechanism that controls the generation of Pol δ3. The underlying importance of this system lies in the altered enzymatic properties of the two forms of Pol δ engendered by the influence of p12. We briefly review how the balance of these two forms is regulated through the degradation of p12. We focus on the roles of Pol δ4, whose cellular functions are less well known. This is significant because recent studies show that this is the form engaged in the homology-dependent repair of double-strand breaks. We consider new horizons for future research into this system and their potential involvement in tumorigenesis.

Keywords: DNA damage response; DNA polymerase δ; DNA repair; cell cycle regulation; homology-directed repair; human DNA replication; tumorigenesis.

Figure 1

The degradation of p12 controls the interplay between Pol δ4 and Pol δ3. The left arm of the diagram gives a concise summary of our previous studies [1,2,3,4,58,59]. The regulated degradation of p12 is the key mechanism for the generation of Pol δ3. These studies also support an essential role for Pol δ3 in lagging-strand DNA synthesis and provide evidence regarding the differential roles of Pol δ4 in HDR. The delineation of the functions of Pol δ4 is still incomplete, and it may serve specific roles in BIR (break-induced replication) and MiDAS (mitotic DNA replication). The question marks are present to indicate that these are important areas for further study, especially regarding their comparative contributions to these processes.

Figure 2

Pol δ4 but not Pol δ3 exhibits stand displacement activity. (A–D). Diagrammatic view of the ability of Pol δ4 and Pol δ3 to perform strand displacement on model oligonucleotide substrates and the processing of Okazaki fragments in concert with Fen 1 and DNA ligase using model oligonucleotide substrates [4,69]. (E). Displacement synthesis in a model D-loop (extension of invading strand). Pol δ4, but not Pol δ3, is predicted to be able to perform D-loop extension [58].

Figure 3

The degradation of p12 in mammalian cells containing Pol δ4 to cells containing Pol δ3 choreographs their participation in DNA repair and replication processes. (A) p12 degradation acts as a mechanism that dictates the two central functions in DNA replication and DNA repair. This diagram provides an overview of the cellular regulation of the degradation of p12, which converts the Pol δ4 tetramer to the Pol δ3 trimer. The removal of p12 leaves behind the Pol δ3 enzyme in vivo via the regulated destruction of the p12 subunit. Thus, cells containing Pol δ4 are converted to cells containing Pol δ3, a phenomenon which occurs under two scenarios. The first is the presence of genotoxic or replication stress (left-hand column). This occurs in an ATR-dependent manner by proteasomal degradation mediated by E3 ubiquitin ligases. Multiple E3 ubiquitin ligases may be involved, including RNF8 and CRL4^Cdt2. This mechanism ensures that Pol δ3 is the operative form of Pol δ activity for gap-filling in excision repair (NER, MMR). The second trigger for p12 degradation (right-hand column) is integrally embedded in the cell cycle regulation of the initiation of DNA synthesis. Here, p12 degradation is ubiquitinylated and targeted for proteasomal degradation by CRL4^Cdt2, which plays a key role in the destruction of licensing factors Cdt1, Set8, and p21 to prevent the re-licensing of the origins. Thus, cells in the S phase contain only Pol δ3, which is the lagging-strand polymerase in DNA replication. The system acts as a flip–flop switch between two cellular states where either Pol δ4 or Pol δ3 is the primary polymerase. This also constrains Pol δ4 from acting when Pol δ3’s function is operative. (B). The p12 subunit binds to the p125 subunit and thereby acts to modulate Pol δ4’s activity. This is of consequence because Pol δ4 possesses functions in DNA transactions that are not shared by Pol δ3. These functions represent “gain of function” attributes that expand the cellular repertoire of Pol δ activity. Notably, Pol δ4, but not Pol δ3, is the primary form engaged in the HDR repair of double-stranded DNA breaks. Thus, the involvement of p12/Pol δ4 in human DNA synthesis in homologous recombination is also subject to control by the p12 regulatory switch shown in (A).