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Eukaryotic Replication Proteins

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    322
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    Eukaryotic replication proteins have functions analogous to those found in bacteria.

    Figure 5.28. Similar structures of processivity factors for DNA replication. The mammalian protein, PCNA (top), is a trimer, each monomer of which has two similar domains. T he trimer forms a circle that surrounds DNA, hence serving as a sliding clamp. The b subunit of DNA polymerase III from E. coli is a dimer (bottom), each monomer of which has three similar domains. These domains have a very similar structure to those of PCNA, despite having only limited sequence similarity. Thus functionally analogous sliding clamps in eukaryotes and prokaryotes have similar structures.

    The template-primer junctions are recognized by the multisubunit replication factor C, or RFC. Like the g complex in E. coli, this enzyme is an ATPase, and it helps to load on the processivity factor PCNA. Thus RFC is carrying out a similar function to the bacterial g-complex.

    One of the first eukaryotic polymerases to be isolated was DNA polymerase a, which is now recognized as a catalyst of primer synthesis. This enzyme contains four polypeptide subunits, one with a polymerase activity (170 kDa), two that comprise a primase activity (50 and 60 kDa), and another subunit of (currently) undetermined function (70 kDa). DNA polymerase a has low processivity but high fidelity. This high fidelity is surprising because no 3' to 5' exonuclease is associated with the enzyme. Polymerase a, possibly with additional primases, catalyzes the synthesis of short segments of DNA and RNA that serve as primers for the replicative polymerases.

    DNA polymerase e is related to polymerase d, and it may play a role in lagging strand synthesis. It is also dependent on PCNA, in vivo. However, no requirement has been identified for it in viral replication systems in vitro.

    The compound aphidicolin will block the growth of mammalian cells. It does this by preventing DNA replication, and the targets of this drug are DNA polymerases a and d (as well as e). The fact that inhibition of these DNA polymerases with aphidicolin also stops DNA replication in mammalian cells argues that indeed, a and d are responsible for replication of nuclear DNA in eukaryotic cells. This conclusion is strongly supported by the phenotype of conditional loss-of-function mutations in the genes encoding the homologs to these polymerases in yeast. Such mutants do not grow at the restrictive temperature, indicating that d and a are the replicative polymerases. The biochemical evidence implicates polymerase a in primer formation, and d appears to be the major polymerases used to synthesize the new strands of DNA.

    Table 5.4: Analogous components of the replication machinery in E. coliand eukaryotic cells.

    Function

    Bacterial (E. coli)

    Number of subunits

    Eukaryotic replication (SV40)

    Number of subunits

    Leading and lagging strand synthesis

    asymmetric dimer, E. colipolymerase III

    10 (3 in core)

    polymerase d

    2

    Sliding clamp

    b subunit

    2

    PCNA

    3

    Clamp loader

    g-complex

    6

    RFC

    multiple

    Primase

    DnaG

    1

    Polymerase a

    4

    Helicase

    DnaB

    6

    T-antigen (SV40)

    6

    Bind single-stranded DNA

    SSB

    1

    RFA

    3

    Swivel

    Gyrase

    4, A2B2

    Topo I

    or Topo II

    1

    2 (homodimer)

    The parallels between bacterial and eukaryotic DNA replication are striking. The overall strategy of synthesis is similar, and analogous proteins carry out similar functions, as listed in Table 5.4. It is difficult to determine whether the proteins carrying out similar functions are actually homologous proteins, i.e. encoded by genes descended from the same gene in the last common ancestor. The protein sequence identities are marginal, and frequently the analogous proteins have different numbers of subunits. These differences complicate the analysis considerably, because different subunits in bacteria or mammals may have similar functions. However, the functional similarities are convincing.

    Several other DNA polymerases have been isolated from eukaryotic cells. DNA polymerase b and e are involved in repair of nuclear DNA. DNA polymerase b is a single polypeptide of 36 kDa, and has no 3' to 5' exonuclease. DNA polymerase g replicates mitochondrial DNA.

    Reverse transcriptaseis frequently referred to as an RNA-dependent DNA polymerase because it can use RNA as a template, but in fact it can use either RNA or DNA as a template. It is encoded by retroviruses, and hence it is present in cells infected with a retrovirus. This enzyme has widespread use in the laboratory for making complementary copies of RNA, called cDNA. Active copies of LINE1 repetitive elements (in mammals) or Ty1 repeats (in yeast), also encode reverse transcriptase. Thus in cells where these retrotransposable elements are being transcribed, active reverse transcriptase is also present. Reverse transcriptase also has an RNase H activity, which will digest away RNA from an RNA-DNA duplex.

    In contrast to the other DNA polymerases discussed in this chapter, terminal deoxynucleotidyl transferasedoes not require a template. It adds dNTPs (as dNMP) to the 3' end of DNA, using that 3' hydroxyl as a primer. It is found in differentiating lymphocytes, and appears to be used physiologically to introduce somatic mutations into immunoglobulin genes. In the laboratory, it is used to add "homopolymer tails" to the ends of DNA molecules by incubating a linear DNA with one particular dNTP and terminal deoxynucleotidyl transferase.

    As will be discussed in more detail in the next chapter, the ends of linear chromosomes (telomeres) must be expanded at each replication or they will eventually become shortened. The enzyme telomerasecatalyzes the addition of many tandem copies of a simple sequence to the ends of the chromosomes. The template for this reaction is an RNA that is a component of the enzyme. Thus telomerase is a reverse transcriptase that only makes copies of the template that it carries, using the 3' end of a chromosomal DNA strand as the primer.


    This page titled Eukaryotic Replication Proteins is shared under a All Rights Reserved (used with permission) license and was authored, remixed, and/or curated by Ross Hardison.

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