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. 2014;13(1):23-31.
doi: 10.4161/cc.27407. Epub 2013 Dec 3.

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

Affiliations

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

Marietta Y W T Lee et al. Cell Cycle. 2014.

Abstract

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.

Keywords: CRL4Cdt2; DNA damage; DNA polymerase δ; DNA replication; RNF8; cell cycle; cell cycle progression; p12 subunit.

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Figures

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Figure 1. Role of RNF8 in targeting of p12 in response to DNA damage and its potential for integrating responses to various genotoxic stimuli. Panel (A) illustrates the role of RNF8 in p12 degradation in generating Pol δ3 at sites of NER, where it fulfils a role in gap-filling after the excision step. Panel (B) shows the special case in S phase cells, where UV damage leads to stalling of the replication fork. Here, Rad18 has been shown to monoubiquitinate PCNA and initiate the process of translesion synthesis. The gray arrows between panels (A andB) indicate the speculative possibility that RNF8 and Rad18 could both participate in the ubiquitination of PCNA and p12. (C) The presence of RNF8 at sites of DSBs suggests that it may participate in the degradation of p12, generating Pol δ3 to function in HR processes. Here, it is also possible that RNF8 may function in the monoubiquitination of PCNA for the recruitment of TLS pols or in the non-canonical polyubiquitination of PCNA for HR processes.
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Figure 2. Regulation of Pol δ during cell cycle progression by the degradation of the p12 subunit. (A) The p12 subunit of Pol δ4 is partially degraded during S phase, leading to the formation of Pol δ3. H1299 cells were synchronized with nocodazole and allowed to progress through the cell cycle after release from nocodazole. p12 levels are shown as solid squares. The data are taken from Figure 4 in Zhang et al. The relative amounts of Pol δ4 and Pol δ3 are shown by the shaded areas (blue for Pol δ4 and pink for Pol δ3). The amounts of Pol δ3 are an approximation based on the loss of p12 and the assumption that Pol δ4 is converted to Pol δ3. The dotted line shows the changes in the levels of p21 in the same experiment. (B) The presence of both Pol δ4 and Pol δ3 during S phase raises questions regarding their functions at the leading and lagging strands at the replication fork. Here we show the possibility that Pol δ3 and Pol δ4 may both be involved in lagging-strand synthesis, and also that Pol δ4 may participate in leading-strand synthesis.
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Figure 3. Okazaki fragment processing by Pol δ4 and Pol δ3. This diagram summarizes the reactions of Pol δ4 and Pol δ3 with Fen1 in the reconstituted system for Okazaki fragment processing. Panel I (blue) shows the operation of the short flap pathway. In reaction “1”, Pol δ extends the primer until it meets a 5′ downstream primer. Pol δ4 performs limited strand displacement to create short flaps of 1–10 nts (“2”) that are cleaved by Fen1 (“3”). This process is iterated to leave a nick that is sealed by DNA ligase. Panel II illustrates the operation of the nick translation pathway. In reaction “1”, Pol δ3 extends the primer until it meets a 5′ downstream primer. Pol δ inserts just 1 or 2 nts (“3”) that represent insertions due to the fraying/dissociation of the 5′ end of the blocking oligonucleotide (strand opening78). Pol δ3 does not perform significant strand displacement. The cleavage of the short flap leads to the formation of mainly 1 nt products. Multiple iterations of reactions “2” and “3” lead to the stepwise movement of the nick 1 nt at a time, viz., a nick translation reaction.

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