Replication Process Manipulation
Replication of genomes is a complex process that involves the interaction of many proteins and enzyme activities. It initiates at specific locations called origin of replication, which are sites of assembly and binding of protein and enzyme complexes termed origin recognition complex (ORC).
The progress and completion of replication is regulated both in space and in time in such a way that it only starts and terminates at the specific origin and termination sites and at predetermined time points of the cell division cycle. The protein and enzyme complexes responsible for the special and temporal control of the three major stages of initiation, elongation, and termination of DNA replication have been characterized in considerable detail. Biochemical, genetic, and in vitro complementation approaches have been used to reveal common sequence and structural features of the replication machinery such as binding sites for replication proteins, DNA unwinding elements, and sites for the attachment to the nuclear matrix and scaffold structures. The detailed mechanisms of how these different elements specify the origin of replication within the whole chromosome is still poorly understood, especially in the mammalian genomes where replication starts at many sites on each chromosome.
Different conditional mutants in bacteria and yeast, which are usually temperature sensitive mutants, have helped to identify the components of the ORC and identify consensus sequences determining the initiation site. DNA methylation of sites in the origin of replication has also been identified as critical in controlling the initiation of replication. Special enzymes called Dam methylases add methyl groups to these sites. During the semiconservative synthesis of DNA, only the old strand is methylated, the new (novel) strand remains non-methylated for a period of time after each replication cycle. The hemimethylated state (i.e., one strand methylated, the other not methylated) of these sites lead to the sequestration of the origin of replication to the bacterial membrane where a membrane bound inhibitor is thought to prevent the binding of the first component of the ORC. Bacterial mutants that lack the Dam methylase (Dam-) are used in recombinant DNA technology. If the copy number of a plasmid vector inside a bacteria needs to be controlled, one introduce this vector in a Dam negative strain and it will only replicate once.
The temporal regulation of the initiation of replication in eukaryotic cells is studied using complementation assays. These are controlled systems where a substrate DNA, is allowed to undergo replication under the control of factors from the cytosol of cells that are at different stages of the cell cycle. Xenopus eggs, which are known to contain replication factors that are sufficient to support several rounds of replication without new protein synthesis, are used to support the replication of DNA in nuclei from another cell type. When a nucleus is introduced into the egg and the nuclear membrane is prevented from disintegrating, the DNA in the injected nucleus will replicate only once. If the nuclear membrane is disrupted, the DNA will undergo several rounds of replication. This indicates the existence of a factor or factors responsible for initiating replication that were depleted from the intact nucleus.
The factors needed to initiate replication, and supplied in this case by the Xenopus cytosol, are called licensing factors. Their depletion from the nucleus after initiation of the first replication prevents further round of replication from occurring. This process, cytosolic licensing can not be imported into the nucleus and are only takes place when the nuclear membrane breaks down during mitosis.
The elongation of replication is maintained by the integrity of the protein and enzyme complex that is formed at the origin of replication. This complex moves along the DNA substrate until it reaches a termination site where it disassembles. The ORC complex in the yeast is an exception and does not disassemble. It is a constitutive chromatin bound complex. The initiation of replication or its inhibition depends, in this case, on the interaction of protein factors, called Cdc6 and Mcm, with the ORC and the origin of replication.
Control of replication termination is very poorly understood in eukaryotic and mammalian systems. The existence of replication termini in plasmids and bacteria was established in the 1980s. As of 2001, however, studies have identified only a few of the number of factors involved in the control of termination in these replicons.
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