The Pol32 Subunit of DNA Polymerase δ Contains Separable Domains for Processive Replication and Proliferating Cell Nuclear Antigen (PCNA) Binding

Abstract

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase ␦ (Pol ␦). Interactions with POL31, the second subunit of Pol ␦, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase ␣, POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol ␦ interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol ␦ complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol ␦ localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.Accurate and efficient DNA replication in eukaryotic cells requires the participation of at least three essential DNA polymerases, Pol ␣, 1 Pol ␦, and Pol ⑀. Several lines of evidence indicate that Pol ␣ and Pol ␦ function in the initiation and elongation of Okazaki fragment synthesis, respectively (reviewed in Refs. 1 and 2). Replication of the C-A-rich strand at telomeres, by nature a lagging strand process, is also dependent on these two DNA polymerases (3). Under special circumstances, e.g. in a yeast mutant carrying a deletion of the Pol ⑀ polymerization domain, Pol ␦ probably also carries out leading strand synthesis (4 -6). However, analyses of exonuclease-deficient DNA polymerase ␦ and ⑀ strains have revealed a strand specificity of mutation rates and spectra that is most easily reconciled with a dominant role for Pol ⑀ in leading strand synthesis (7-9).Pol ␦ from Saccharomyces cerevisiae consists of three subunits of 125, 55, and 40 kDa (10). An additional small fourth subunit is found in the enzymes from Schizosaccharomyces pombe and human, (11-13). Deletion of the Cdm1 gene for the fourth subunit in S. pombe is genetically silent in the absence of additional mutations in Cdc1 encoding the second subunit (14). However, efficient overproduction of soluble fission yeast Pol ␦ in baculovirus required the presence of Cdm1 (15). The analogous p12 human subunit could be reconstituted with the human three-subunit Pol ␦ into a stable four-subunit enzyme, and significantly stimulated its activity (13).The catalytic subunit and the second subunit, encoded in S. cerevisiae by the POL3 and POL31 (HYS2) genes, respectively, are highly conserved in eukaryotes. The genes are also...

Evidence Suggesting that Pif1 Helicase Functions in DNA Replication with the Dna2 Helicase/Nuclease and DNA Polymerase δ

“…If Pif1 helicase creates 5Ј flaps via its 5Ј-to-3Ј helicase activity, then deletion of Pif1 should lead to reduced occurrence of 5Ј flaps in the cell, which might, in part, account for the reduced re- on May 10, 2018 by guest http://mcb.asm.org/ quirement for the 5Ј flap helicase/nuclease activity of Dna2 in a pif1⌬ mutant. POL32 is a subunit of pol ␦ that leads to decreased 5Ј flap strand displacement by pol ␦ when mutated (25). Since deletion of POL32 also suppresses dna2-1 temperature sensitivity (11), we asked whether deleting POL32 could suppress the residual temperature-sensitive growth of the dna2⌬ pif1⌬ double mutant.…”

“…First, the temperature sensitivity of dna2-1 mutants and the DNA damage sensitivity of dna2-2 mutants, but not the lethality of dna2⌬, are suppressed by the deletion of POL32 (11). The POL32 gene encodes a subunit of pol ␦ that is required for optimum processivity, and its deletion can be expected to reduce strand displacement in vivo as it has been demonstrated to do in vitro (12,25). This would explain the reduced requirement for DNA2.…”

Regulation of polymerase exchange between Polη and Polδ by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme

“…This distinction may be understood in view of the structural difference between yeast Pol␦ and the T4 gp43. Although the processivity factors PCNA and gp45 are highly similar in their 3D structures (both are toroids with a diameter of Ϸ60 Å), the yeast Pol␦ (220 kDa) is twice as large as gp43 (100 kDa) and probably has more sites of interactions with its cognate clamp protein than the T4 Pol (24). The bulkiness of Pol␦ may exclude the simultaneous binding of two copies of Pol␦ to PCNA, which could be an essential intermediate for the active Pol exchange process as suggested by a previous study (9).…”

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

“…Further supporting this conclusion, deletion of POL32, which encodes the subunit of pol ␦ that interacts with PCNA, rescued the temperature sensitivity of the dna2⌬pif1⌬ double mutant (12,27). Importantly, pol ␦ exhibited reduced strand displacement activity when POL32 was deleted (12,28,29). The combination of pif1⌬ and pol32⌬ is believed to create a situation in which virtually no long flaps are formed, eliminating the requirement for Dna2 flap cleavage (27).…”