Distinctive activities of DNA polymerases during human DNA replication

Abstract

DNA polymerases (pols) have a central role in DNA replication and maintenance of chromosomal DNA [1]. At least 14 pols have been identified in the mammalian cell, but only three -pols a, d and e -are needed to synthesize the bulk of DNA during nuclear DNA replication. These pols are structurally related, belonging to the family B DNA polymerases [2]. Nonetheless, all three perform additional roles in other DNA transactions as well as transduce signals of cell cycle control and DNA damage response [1].Only pol a is capable of initiating DNA synthesis de novo owing to its associated primase activity [3]. The major function of pol a ⁄ primase is synthesizing a short RNA-DNA primer of 30-40 nucleotides that serves both to initiate leading strand DNA replication and to provide precursors of the 200 nucleotide-long Okazaki fragments on the lagging strand [4][5][6]. Pol a ⁄ primase is then replaced by the elongating pols d or e. This switch from pol a to pol d is controlled by replication factor C, which loads the processivity factor, Keywords cell cycle; DNA polymerase; DNA replication; electron microscopy; UV crosslinking The contributions of human DNA polymerases (pols) a, d and e during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol d was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols a and e remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol d, unlike pols a and e, increased in the late S phase. Moreover, pol d neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol e were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol e to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol d was mainly dispersed on fibrous structures. Pol a and proliferating cell nuclear antigen displayed partial colocalization with pol d and e, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols d and e pursue their functions at least partly independently during DNA replication.Abbreviations BrdU, bromodeoxyuridine; CLSM, confocal laser-scanning microscopy; EM, electron microscopy; immuno-EM, immuno electron microscopy; MCM2, minichromosome maintenance 2; NP-40, Nonidet P-40; PCNA, proliferating cell nuclear antigen; pol, DNA polymerase.

DNA polymerase ε and δ proofreading suppress discrete mutator and cancer phenotypes in mice

“…Studies in yeast show that Pols and ␦ synthesize and proofread opposite DNA strands at defined replication origins (10,(14)(15)(16)(17)(18). However, Pols and ␦ also may act independently (33,34), and some replication forks may involve only 1 proofreading polymerase (14). Considered together, our results suggest a model in which Pol and Pol ␦ replicate and proofread different regions of the mouse genome encoding tissue-specific genes that suppress cancer or are required for embryogenesis.…”

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

“…86 In human cells, studies of the localization of Pol δ and Pol ε as well as analysis of their association with chromatin and lamins have suggested that they may act partly independently during S phase progression. [87][88][89] While Pol δ3 might be excluded as a player in leading-strand synthesis, the idea that Pol δ4 could participate in leading-strand synthesis is still an open possibility. Whether and why both Pol δ3 and Pol δ4 participate in lagging-strand synthesis remain questions to be answered.…”

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

“…A study using different experimental approaches to examine the association of Pol d, Pol e, and Pol a at different stages of the S phase in HeLa cells has provided evidence that both Pol e and Pol d participate in chromosomal DNA replication and that Pol e plays a Environmental and Molecular Mutagenesis. DOI 10.1002/em greater role in early S phase while Pol d plays a greater role in late S phase [Rytkonen et al, 2006].…”