Eukaryotes DNA synthesis

Eukaryotic chromosomes, unlike their bacterial counterparts, are linear rather than- circular. Since RNA oligonucleotides prime both prokaryotic and eukaryotic DNA synthesis, the 5 termini of the daughter... 

Consequently, approximately one month is required for this DNA replication. Eukaryotic DNA synthesis is significantly faster than expected because each chromosome contains multiple replication units (replicons). 

The mechanism of termination of DNA synthesis is not clear. Whether these same mechanisms apply to eukaryotic DNA synthesis is not yet known. 

RNA primers are involved in eukaryotic DNA synthesis (Komberg, 1976 Edenberg and Huberman, 1975). DNA polymerases are unable to initiate DNA synthesis directly on a DNA template (Weissbach, 1977). In prokaryotes it has been shown that an RNA segment consisting of 40-50 nucleotide residues, copied from a single-stranded DNA template, serves as the primer for attachment of deoxyribonucleotides at the 3 end (Dressier, 1975). The primer RNA is later cleaved by a specific ribonuclease. 

The eukaryotic somatic cell cycle is defined by a sequential order of tasks a dividing cell has to complete it must replicate its DNA, segregate its chromosomes, grow, and divide. The cell cycle can be divided into four discrete phases. DNA replication is restricted to S phase (DNA synthesis phase), which is preceded by a gap phase called G1 and followed by a gap phase called G2. During mitosis (M phase) the sister chromatids are segregated into two new daughter nuclei and mitosis is completed by the division of the cytoplasm termed cytokinesis (Fig. 1). 

Figure 36-16. The discontinuous poiymerization of deoxyribonucleotides on the lagging strand formation of Okazaki fragments during iagging strand DNA synthesis is illustrated. Okazaki fragments are 100-250 nt iong in eukaryotes, 1000-2000 bp in prokaryotes.

Figure 1. The cell cycle as a Cdc2 cycle. Progression through the eukaryotic cell cycle is sensitive to the phosphorylation state of Cdc2. A block to DNA synthesis (S) prevents dephosphorylation, and hence activation, of Cdc2. Impaired spindle function will prevent deactivation of Cdc2 and thus blocks exit from M phase (Hoyt et al., 1991 Li and Murray, 1991 reviewed in Nurse, 1991). Exit from M phase requires destruction of the regulatory subunit, Cyc B. Dephosphorylation of Cdc2 at thr-161 may act to destabilize the Cdc2/Cyc B complex and thus allow the ubiquitination of Cyc B followed by its destruction.

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. 

Eukaryotic organisms HeLa cells DNA synthesis inhibition - - Painter and Howard 1982 KCN... 

Variants of histone H2A are most common in higher eukaryotes. Thus far, five H2A-type histones have been described, of which two are found in all eukaryotes from yeast to mammals (Table 1). These are the histones H2A.X, and H2A.Z (Thatcher and Gorovsky 1994). While all other eukaryotes possess a canonical H2A, S. cerevisiae utilizes H2A.X as general, replication-dependent H2A form. Vertebrates possess an additional H2A variant named macroH2A, while the fifth known H2A variant H2ABBd (Barr body-deficient), is only conserved for mammals (Chow and Brown 2003 Gautier et al. 2004). Besides the most abundant canonical H2A, which is deposited into chromatin during DNA synthesis, other H2A variants also are synthesized outside of the S phase. Like specialized variants of H3, these proteins also are available for incorporation into chromatin independent of DNA replication. 

In higher eukaryotes, at the onset of S phase cyclin A accumulates which stimulates DNA synthesis. The amount of cyclin A continues to be high after the S phase because of its role in chromosome condensation. Cyclin A is degraded when cells enter prometaphase. The level of another cyclin called cyclin B rises during G2 phase, which helps to complete the chromosome condensation and spindle assembly, which allow transition to metaphase. Cyclin B is degraded by APC during metaphase. ... 

Kandell and Bernstein published one of the earliest reports to suggest that bile acids also demonstrate DNA-damaging effects in eukaryotic cells. They showed that human foreskin fibroblasts underwent unscheduled DNA synthesis (indicating DNA repair), as measured by tritiated thymidine incorporation when cells were treated with increasing concentrations of sodium deoxycholate or chenodeoxycholate. Utilising mutant Chinese hamster ovary cells deficient in strand rejoining (EM9), the authors were able to demonstrate that the repair of deoxycholate-induced DNA damage was dependent on strand break repair capacity. 

In bacteria, error-prone translesion DNA synthesis, involving TLS DNA polymerases, occurs in response to very heavy DNA damage. In eukaryotes, similar polymerases have specialized roles in DNA repair that minimize the introduction of mutations. 

The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been discussed. Eukaryotic single-stranded DNA-binding proteins and ATP-dependent DNA helicases have been identified, whose functions are analogous to those of the prokaryotic enzymes previously discussed. In contrast, RNA primers are removed by RNase H. 

There are at least five classes of eukaryotic DNA polymerases. Pol a is a multisubunit enzyme, one subunit of which performs the primase function. Pol a 5 ->3 polymerase activity adds a short piece of DNA to the RNA primer. Pol 8 completes DNA synthesis on the leading strand and elongates each lagging strand fragment, using 3 ->5 exonuclease activity to proofread the newly synthesized DNA. Pol p and pol e are involved in carrying out DNA "repair," and pol y replicates mitochondrial DNA. 

Since eukaryotic chromosomes are linear, the ends of these chromosomes require a special solution to ensure complete replication. This can be seen in figure 26.26. At the very end of a linear duplex a primer is necessary to initiate DNA replication. After RNA primer removal there is bound to be a gap at the 5 end of the newly synthesized DNA chains. Since DNA synthesis always requires a primer the usual way of filling this gap is not going to solve the problem. This dilemma is overcome by a special structure at the ends (telomeres) of eukaryotic chromosomes and a special type of reverse transcriptase (telomerase) that synthesizes telomeric DNA. In many eukaryotes the telomeres contain short sequences (frequently hexamers) that are tan-demly repeated many times. Telomerase contains an RNA that binds to the 3 ends and also serves as a template for the extension of these ends. Prior to replication, the 3 ends of the chromosome are extended with additional tandemly repeated hexamers. The 3 ends are extended sufficiently so that there is room to accommodate an RNA primer. In this way there is no net loss of DNA from the 5 ends as a result of replication. After replication the 3 end is somewhat... 

Synthesis at the ends of a eukaryotic chromosome. One end of the linear DNA of a eukaryotic chromosome is diagrammed. A flush-ended DNA duplex presents a problem for completing synthesis at the 5 end (a). This is because of the RNA primer requirement for DNA synthesis. When the primer at the 5 end is removed there is no conventional way to fill the gap. A solution to this problem is shown in (b). The ends of eukaryotic chromosomal DNAs consist of highly repetitious tandem repeats (telomeres). These repeats on the 3 end serve as both primer and template for extending the 3 end. The extended 3 end can accommodate a primer RNA, so after chromosomal DNA replication no loss occurs from the 5 end of the DNA. Another process is needed to remove the extension from the 3 end. New synthesis is indicated in red. The zigzag represents primer. 

Eukaryotic DNA is replicated at a slower rate than prokaryotic DNA. One reason may be the requirement for the deposition of histone proteins on DNA (histone synthesis and DNA replication are coupled). Describe a model for the replication of eukaryotic DNA and nucleosome formation. 

In prokaryotes DNA, RNA, and protein synthesis all take place in the same cellular compartment. In eukaryotes the DNA is compartmentalized in the cell nucleus, and it became clear long before the biochemistry of these three processes was understood that DNA synthesis takes place in the nucleus, whereas the bulk of protein synthesis takes place in the cytoplasm. From these observations on eukaryotes it was self-evident that DNA cannot be directly involved in the synthesis of protein but must somehow transmit its genetic information for protein synthesis to the cytoplasm. Careful experiments with radioactive labels were used to demonstrate that RNA synthesis takes place in the nucleus much of this RNA is degraded rather quickly, but the portion that survives is mostly transferred to the cytoplasm (fig. 28.1). From observations of this kind it became clear that RNA was the prime candidate for the carrier of genetic information for the synthesis of proteins. 

DNA in eukaryotic chromosomes is complexed with histone proteins in complexes called nucleosomes. These DNA-protein complexes are disassembled directly in front of the replication fork. The nucleosome disassembly may be rate-limiting for the migration of the replication forks, as the rate of migration is slower in eukaryotes than prokaryotes. The length of Okazaki fragments is also similar to the size of the DNA between nucleosomes (about 200 bp). One model that would allow the synthesis of new eukaryotic DNA and nucleosome formation would be the disassembly of the histones in front of the replication fork and then the reassembly of the histones on the two duplex strands. Histone synthesis is closely coupled to DNA replication. 

Okazaki fragment. A short segment of single-stranded DNA that is an intermediate in DNA synthesis. In bacteria, Okazaki fragments are 1,000-2,000 bases in length in eukaryotes, 100-200 bases in length. 

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