Replitase: Complete machinery for DNA synthesis

Abstract

Replication of nuclear DNA in eukaryotes presents a tremendous challenge, not only due to the size and complexity of the genome, but also because of the time constraint imposed by a limited duration of S phase during which the entire genome has to be duplicated accurately and only once per cell division cycle. A challenge of this magnitude can only be met by the close coupling of DNA precursor synthesis to replication. Prokaryotic systems provide evidence for multienzyme and multiprotein complexes involved in DNA precursor synthesis and DNA replication. In addition, fractionation of nuclear proteins from proliferating mammalian cells shows co-sedimentation of enzymes involved in DNA replication with those required for synthesis of deoxynucleoside triphosphates (dNTPs). Such complexes can be isolated only from cells that are in S phase, but not from cells in G(0)/G(1) phases of cell cycle. The kinetics of deoxynucleotide metabolism supporting DNA replication in intact and permeabilized cells reveals close coupling and allosteric interaction between the enzymes of dNTP synthesis and DNA replication. These interactions contribute to channeling and compartmentation of deoxynucleotides in the microvicinity of DNA replication. A multienzyme and multiprotein megacomplex with these unique properties is called "replitase." In this article, we summarize some of the relevant evidence to date that supports the concept of replitase in mammalian cells, which originated from the observations in Dr. Pardee's laboratory. In addition, we show that androgen receptor (AR), which plays a critical role in proliferation and viability of prostate cancer cells, is associated with replitase, and that identification of constituents of replitase in androgen-dependent versus androgen-independent prostate cancer cells may provide insights into androgen-regulated events that control proliferation of prostate cancer cells and potentially offer an effective strategy for the treatment of prostate cancer.

Metastasis Suppressor NM23-H1 Promotes Repair of UV-Induced DNA Damage and Suppresses UV-Induced Melanomagenesis

“…The rapid co-localization of NM23-H1 with (6-4) photoproduct lesions raises the possibility that enzymes involved in dNTP synthesis are functionally coupled with DNA repair components at sites of DNA damage (39-41). Indeed, recent studies have implicated deoxyribonucleotide metabolizing enzymes in the DNA repair response, such as ribonucleotide reductase, p53R2 and thymidine kinase (42-45).…”

“…Indeed, recent studies have implicated deoxyribonucleotide metabolizing enzymes in the DNA repair response, such as ribonucleotide reductase, p53R2 and thymidine kinase (42-45). The possibility remains open that the NDPK activity may deliver nucleotides directly to the DNA machinery during DNA repair processes, as hypothesized by multiple investigators (39-41). …”

Androgen Receptor Interacts with Telomeric Proteins in Prostate Cancer Cells

“…AR has been shown to be associated with enzymes of DNA synthesis (28), with cell cycle regulatory proteins required for the transition of cells from G 1 to S phase (28), and with some of the DNA doublestrand break repair proteins such as Ku70 and Ku80 (29), which also interact with TRF1 and TRF2 in telomeric complexes (8,9). Mechanistically, whereas AR binds to DNA in its role as a transcription factor, AR appears to interact with proteins to exert its influence on DNA replication and repair.…”

“…Besides its classical role as a transcription factor, there is accumulating evidence that AR has a role in DNA replication (27,28), repair (29), and recombination (30). AR has been shown to be associated with enzymes of DNA synthesis (28), with cell cycle regulatory proteins required for the transition of cells from G 1 to S phase (28), and with some of the DNA doublestrand break repair proteins such as Ku70 and Ku80 (29), which also interact with TRF1 and TRF2 in telomeric complexes (8,9).…”

Targeting nuclear thymidylate biosynthesis

“…However, in the 1980s, there were reports demonstrating that TYMS could also be localized to the nucleus. Thereafter, co-sedimentation and co-fractionation experiments in mammalian cells indicated that some, but not all, of the enzymes involved in deoxynucleotide biosynthesis were found to co-localize in a multiprotein complex with the DNA replication machinery, and this complex was referred to as the “replitase” 87-89 . Interestingly, the nuclear localization of TYMS was not observed in yeast, rather S. cerevesiae TYMS was found to localize to the nuclear periphery 90 .…”