Such studies indicate that yeast mutants lacking any of these three DNA polymerases are unable to proliferate, implying a critical role for polymerase ε as well as for α and δ. Second, polymerases α, δ, and ε are found in yeasts as well as in mammalian cells, enabling the use of the powerful approaches of yeast genetics (see Chapter 3) to test their biological roles directly. The ability to study replication in vitro has allowed direct identification of the enzymes involved, and analysis of such cell-free systems has shown that polymerases α and δ are required for SV40 DNA replication. First, replication of the DNAs of some animal viruses, such as SV40, can be studied in cell-free extracts. Two types of experiments have provided further evidence addressing the roles of polymerases α, δ, and ε in DNA replication. In contrast, polymerase β is active in nondividing and dividing cells, suggesting that it may function primarily in the repair of DNA damage. ![]() Polymerases α, δ, and ε are most active in dividing cells, suggesting that they function in replication. The other four enzymes are located in the nucleus and are therefore candidates for involvement in nuclear DNA replication. Polymerase γ is located in mitochondria and is responsible for replication of mitochondrial DNA. coli DNA thus involves two distinct DNA polymerases, the specific roles of which are discussed below.Įukaryotic cells contain five DNA polymerases: α, β, γ, δ, and ε. The original polymerase I mutant was not completely defective in that enzyme, and later experiments showed that the residual polymerase I activity in this strain plays a key role in the replication process. It is now known that, in addition to polymerase III, polymerase I is also required for replication of E. Temperature-sensitive polymerase III mutants, however, were unable to replicate their DNA at high temperature, and subsequent studies have confirmed that polymerase III is the major replicative enzyme in E. coli with mutations in polymerase II were found to grow and otherwise behave normally, so the role of this enzyme in the cell is unknown. The potential roles of these enzymes were investigated by the isolation of appropriate mutants. ![]() coli must contain other DNA polymerases, and subsequent experiments led to the identification of two such enzymes, now called DNA polymerases II and III. The conclusion that polymerase I is not required for replication implied that E. Several thousand colonies were then cultured and screened to identify (more.) coli was treated with a chemical mutagen, and individual bacterial colonies were isolated by growth on semisolid medium. Isolation of a mutant deficient in polymerase I. On the other hand, the mutant bacteria were extremely sensitive to agents that damage DNA (e.g., ultraviolet light), suggesting that polymerase I is involved primarily in the repair of DNA damage rather than in DNA replication per se. Surprisingly, the mutant bacteria grew normally, leading to the conclusion that polymerase I is not required for DNA replication. Analysis of a few thousand colonies led to the isolation of the desired mutant, which was almost totally defective in polymerase I activity. coli were treated with a chemical (a mutagen) that induces a high frequency of mutations, and individual bacterial colonies were isolated and screened to identify a mutant strain lacking polymerase I. ![]() ![]() coli that was deficient in polymerase I ( Figure 5.1). The multiplicity of DNA polymerases was first revealed by the isolation of a mutant strain of E. Instead, it is now clear that both prokaryotic and eukaryotic cells contain several different DNA polymerases that play distinct roles in the replication and repair of DNA. Ironically, however, this first DNA polymerase to be identified (now called DNA polymerase I) is not the major enzyme responsible for E. The ability of this enzyme to accurately copy a DNA template provided a biochemical basis for the mode of DNA replication that was initially proposed by Watson and Crick, so its isolation represented a landmark discovery in molecular biology. DNA polymerase was first identified in lysates of E.
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