THE REPLICATION COMPLEX

Enhanced tolerance to DNA adducts has been fonnd in some cisplatin-resistant ceUs. Mechanisms that are involved are enhanced postreplicative bypass the ability of the replication complex to synthesize DNA downstream a cisplatin-indnced lesion and defects in the repair process named mismatch repair (MMR). 

Eventually, the DNA would become so tightly wound that movement of the replication complex would be energetically impossible. 

Errors that occur during replication are corrected by enzymes associated with the replication complex. 

Only a few substances act directly on DNA polymerases they often are effective only on one or a small number of polymerases. For example, acyclovir inhibits the DNA polymerase of herpes simplex. Aphidicolin inhibits pol a, pol S (but not pol j8 or pol y), many viral polymerases, and pol I and pol II of yeast this compound is extremely valuable in laboratory research on DNA replication. Other components of the replication complex can also be inhibited for example, 2 -dideoxyazidocytidine is an inhibitor of bacterial primase, and coumermycin, novobiocin, oxolinic acid, and nalidixic acid are effective inhibitors of DNA gyrase in bacteria. 

In many recombination pathways, a DNA molecule with a free end recombines with a DNA molecule having no free ends available for interaction. DNA molecules with free ends are the common result of double-stranded DNA breaks, but they may also be generated in DMA replication if the replication complex stalls. This type of recombination has been studied extensively in E. coli, but it also takes place in other organisms through the action of proteins homologous to those of E. a)[i. Often dozens of proteins participate in the complete recombination process. However, the key protein is RecA, another member of the AAA... 

Primosome - a complex containing a primase and helicase. It helps to initiate DNA replication by synthesizing an RNA primer and to elongate it by unwinding the strands in advance of the replication complex. 

Enzymes - Eukaryotic cells contain five DNA polymerases. Three of them (polymerases oi, A, and s) are used during S phase replication. Table 24.2 and Table 24.3 describe the properties of eukaryotic and prokaryotic DNA polymerases. As in prokaryotes, the replication complex also contains other proteins, including helicases and a number of accessory proteins called replication factors. 

F. 13.9. Replication complex in eukaryotes. The lagging strand is shown looped around the replication complex. Single-strand binding proteins (not shown) are bound to the unpaired, single-stranded DNA. Other proteins also participate in this complex (see text). 

Additional activities that occur during replication include proofreading and DNA repair. Pol 8, which is part of the replication complex, has the 3 5 -exonuclease activity required for proofreading. Enzymes that catalyze repair of mismatched bases are also present (see section III.B.3 of this chapter). Consequently, eukaryotic replication occurs with high fidelity approximately one mis-pairing occurs for every 10 to 10 nucleotides incorporated into growing DNA chains. 

As a general rule, novo initiation of ENA synthesis vivo occurs with purine rather than pyrimidine triphosphates. pUp is therefore an unlikely terminus for nascent viral ENA. This consideration led us to suggest that the pUp terminus of mRNA arose from the 5 end of viral ENA by processing (e.g. cleavage of a primer) as the newly made ENA is released from the replication complex (5). Evidence that virion ENA has a 5 terminus distinct from that of mRNA has been obtained as follows ... 

Replication scheme of poliovirus with emphasis on possible functions of VPg. Note that the 5 ends of the template strands in the replication complex are shown to be VPg-linked this may not be necessary. 

Viral ERA synthesis in the picornavirus group is associated with membrane structirres (25, 26) and can be isolated in the form of a replication complex by centrifugation of the cytoplasmic extracts at 20,000 g (27). The phosphorylase activity was shown to be associated with the replication complexes (Table 7)> result expected, as dsRRA structures were in the P-20 fraction. 

The ribonuclease activity was found in a sedimentable fraction known to contain the replication complex where synthesis of virus-coded MA takes place. Therefore, the antiviral action of interferon may be explained in this particular system by the ability of such a nuclease to degrade viral mRNA. Although the nuclease is not only active on mengo virus ENA its location within the cell would limit its action to the virus-coded single stranded ENA, the final consequence being some discrimination between viral and host cell messengers. This finding does not exclude the possibility that the inhibition of protein synthesis also occurs by other mechanisms such as impaired initiation of translation. 

Synthesis of the viral EUA occurs exclusively in the cytoplasm of infected cells (8, 9), namely in a membrane-bound structure known as the replication complex (section Y). 

As an oligo(A)-primed, terminal adenylate transferase of cellular origin was found in tight association with the same membranes the replication complex is bound to, the longer poly(A) stretches were thought to result from either a mechanism of "slippage" or by end addition of adenylate residues (89). 

All viral structures involved in picornavirus RNA synthesis are tightly associated with the smooth cytoplasmic membranes (50 Synthesis of viral RNA occurs exclusively in the replication complex (RC), a complex structure including the RNA template and a virus-coded RNA polymerase (52). 

The replication complex can be isolated with the particulate fraction of the cytoplasm of picornavirus-infected cells (71) Under proper conditions the ECs are able to continue vitro the synthesis of ENA, providing a unique tool to study the mechanism of ENA synthesis with all the advantages (and all the limitations, too) of an m vitro cell-free system. 

DnaB is the key heUcase for replication of the genome E. coli. However, other helicases such as Rep and PriA are also involved in replication and interact with other components of the replication complex called the replisome. 

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