DNA Replication:- Replication in Eukaryotic Cells Is More Complex
The DNA molecules in eukaryotic cells are considerably larger than those in bacteria and are organized into com plex nucleoprotein structures . The essential features of DNA replication are the same in eukaryotes and prokaryotes, and many of the protein complexes are functionally and structurally conserved. However, some interesting variations on the general principles discussed above promise new insights into the regulation of replication and its link with the cell cycle.
Origins of replication, called autonomously replicating sequences (ARS) or replicators, have been identified and best studied in yeast. Yeast replicators span ~150 bp and contain several essential conserved sequences. About 400 replicators are distributed among the 16 chromosomes in a haploid yeast genome. Initiation of replication in all eukaryotes requires a multi subunit protein, the origin recognition complex (ORC), which binds to several sequences within the replicator. ORC interacts with and is regulated by a number of other proteins involved in control of the eukaryotic cell cycle. Two other proteins, CDC6 (discovered in a screen for genes affecting the cell division cycle) and CDT1 (Cdc10-dependent transcript 1), bind to ORC and mediate the loading of a heterohexamer of minichromo some maintenance proteins (MCM2 to MCM7). The MCM complex is a ring-shaped replicative helicase, analogous to the bacterial DnaB helicase. The CDC6 and CDT1 proteins have a role comparable to that of the bacterial DnaC protein, loading the MCM helicase onto the DNA near the replication origin.
The rate of replication fork movement in eukaryotes (~50 nucleotides/s) is only one-twentieth that ob served in E. coli. At this rate, replication of an average human chromosome proceeding from a single origin would take more than 500 hours. Replication of human chromosomes in fact proceeds bidirectionally from many origins, spaced 30,000 to 300,000 bp apart. Eu karyotic chromosomes are almost always much larger than bacterial chromosomes, so multiple origins are probably a universal feature in eukaryotic cells. Like bacteria, eukaryotes have several types of DNA polymerases. Some have been linked to particular functions, such as the replication of mitochondrial DNA. The replication of nuclear chromosomes involves DNA polymerase α, in association with DNA polymerase δ. DNA polymerase α is typically a multisubunit enzyme with similar structure and properties in all eukaryotic cells. One subunit has a primase activity, and the largest subunit (Mr ~180,000) contains the poly merization activity. However, this polymerase has no proofreading 3n5 exonuclease activity, making it unsuitable for high-fidelity DNA replication. DNA polymerase is believed to function only in the synthesis of short primers (containing either RNA or DNA) for Okazaki fragments on the lagging strand. These primers are then extended by the multisubunit DNA polymerase δ. This enzyme is associated with and stimulated by a protein called proliferating cell nuclear anti gen (PCNA; Mr 29,000), found in large amounts in the nuclei of proliferating cells. The three-dimensional structure of PCNA is remarkably similar to that of the β subunit of E. coli DNA polymerase III , although primary sequence homology is not evident. PCNA has a function analogous to that of the β subunit, forming a circular clamp that greatly enhances the processivity of the polymerase. DNA polymerase has a 3→5 proofreading exonuclease activity and appears to carry out both leading and lagging strand synthesis in a complex comparable to the dimeric bacterial DNA polymerase III.
Yet another polymerase, DNA polymerase ε, re places DNA polymerase in some situations, such as in DNA repair. DNA polymerase may also function at the replication fork, perhaps playing a role analogous to that of the bacterial DNA polymerase I, removing the primers of Okazaki fragments on the lagging strand.
Many DNA viruses encode their own DNA polymerases, and some of these have become targets for pharmaceuticals. For example, the DNA polymerase of the herpes simplex virus is inhibited by acyclovir, a com pound developed by Gertrude Elion . Acyclovir consists of guanine attached to an incomplete ribose ring. It is phosphorylated by a virally encoded thymidine kinase; acyclovir binds to this viral enzyme with an affinity 200-fold greater than its binding to the cellular thymidine kinase. This ensures that phosphorylation occurs mainly in virus-infected cells. Cellular kinases con vert the resulting acyclo-GMP to acyclo-GTP, which is both an inhibitor and a substrate of DNA polymerases, and which competitively inhibits the herpes DNA polymerase more strongly than cellular DNA polymerases. Because it lacks a 3 hydroxyl, acyclo-GTP also acts as a chain terminator when incorporated into DNA. Thus viral replication is inhibited at several steps.
Two other protein complexes also function in eu karyotic DNA replication. RPA (replication protein A) is a eukaryotic single-stranded DNA–binding protein, equivalent in function to the E. coli SSB protein. RFC (replication factor C) is a clamp loader for PCNA and facilitates the assembly of active replication complexes. The subunits of the RFC complex have significant sequence similarity to the subunits of the bacterial clamp loading (γ) complex.
The termination of replication on linear eukaryotic chromosomes involves the synthesis of special structures called telomeresat the ends of each chromo some, as discussed in the next chapter.
