Stages of DNA Synthesis

coli cells, DNA replication starts at a specific site called oriC. The oriC locus contains only 245 base pairs. Similar sequences are responsible for initiating the synthesis of plasmid and bacteriophage DNA. The oriC nucleotide sequence binds several units of the tetrameric form of the dnaA protein. This protein is named for the gene that encodes it. The dnaB and dnaC proteins then bind to the complex. As a result of binding these proteins, a portion of the helical DNA is unwound. This forces the rest of the DNA into a left-handed double helix that wraps around the proteins to give a structure 

All DNA polymerases add mononucleotides to the 3 end of an existing primer. Consequently a special primer is needed for DNA to replicate in its entirety. RNA polymerases can initiate polymer synthesis without a primer thus short RNA primers are used to initiate DNA synthesis. 

The RNA oligonucleotides are complementary to a sequence on one of the strands of the DNA template and base pair with a portion of the DNA molecule. Subsequently, deoxyribonucleotides are covalently attached to the RNA primer. The synthesis of the primer itself is catalyzed by a special RNA polymerase called primase. Similar RNA polymerase-like enzymes are used to prime the synthesis of certain viral DNAs and eukaryotic DNA. 

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Under the above conditions, the radioactive dATP present in the first stage of DNA synthesis is the limiting component of the reaction. Depending on the amount of template DNA present, primers may be extended for only a short length. The addition of nonradioactive dATP during the second stage of extension minimizes this problem. 

The antifungal agent 5-fluorocytosine also interferes with these early stages of DNA synthesis. Through conversion to the nucleoside triphosphate it subsequently blocks thymidylic acid production through inhibition of the enzyme thymidylate synthetase (Fig. 12.6). 

This is a precisely choreographed process. Each stage involves complex regulatory mechanisms to ensure that progress through the cell cycle is accomplished one step at a time, is unidirectional, and permits exactly one round of DNA synthesis, resulting in precise duplication of the cellular chromosomes. It is a beautiful, if complex and not yet fully complete, story. 

It is well established that actinomycin D inhibits DNA-directed RNA synthesis by binding to guanosyl residues in the DNA molecule. This disrupts the transcription of genetic information and thereby interferes with the production of essential proteins. DNA synthesis may also be inhibited, being reduced by 30% to 40% in utero. It is clear that in the initial stages of embryogenesis, synthesis of RNA for protein production is vital, and it is not surprising that inhibition of this process may be lethal. 

The first advance came with the visualization of DNA synthesis immunohis-tochemically using bromodeoxyuridine (BrdU) (Miller and Nowakowski 1988). The second significant advance was the identification of new markers that allowed neurons to be distinguished from glia and that more precisely determined the developmental stage of cell populations (reviewed by Pevny and Rao 2003). These... 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

Georgellis A, Parvinen M, Rydstrom J. 1989. Inhibition of stage-specific DNA synthesis in rat spermatogenic cells by polycyclic aromatic hydrocarbons. Chem Biol Interact 72(1-2) 79-92. 

Qiao, D., Seidlcr, F. J., Violin, J. D., and Slotkin, T. A. (2003b). Nicotine is a developmental neurotoxicant and neuroprotcc-tam Stage-selective inhibition of DNA synthesis coincident with shielding from effects of chlorpyrifos. Dev. Brain Res. 147, 183-190. 

Figure 8.1 The cell cycle. Cells pass through four stages of growth during each mitotic cycle. Cells that contain a double complement ofDNA (G cells) divide during mitosis (M phase). Following a "gap" (G phase) cells may either differentiate and remain out of the cycle for a long period of time (G period) or begin the process of DNA synthesis (Sphase). Another gap (Gf) follows.

Reflect and t ply What is the relationship between control of DNA synthesis in eukaryotes and the stages of the cell cycle ... 

Synchronized cultures of HeLa cells display fluctuations in levels of thy-midylate kinase activity that are related to stages of the mitotic cycle thus, thymidylate kinase activity rises at the time of DNA synthesis and falls during division (41). 

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