Chapter 12 The Replicon: Initiation of Replication

12.1 Introduction replicon – A unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication. origin – A sequence of DNA at which replication is initiated.

12.1 Introduction single-copy replication control – A control system in which there is only one copy of a replicon per unit bacterium. The bacterial chromosome and some plasmids have this type of regulation. multicopy replication control – Replication occurs when the control system allows the plasmid to exist in more than one copy per individual bacterial cell.

12.2 An Origin Usually Initiates Bidirectional Replication semiconservative replication – Replication accomplished by separation of the strands of a parental duplex, with each strand then acting as a template for synthesis of a complementary strand. A replicated region appears as a replication bubble within nonreplicated DNA. Figure 12.01: Replicated DNA is seen as a replication bubble flanked by nonreplicated DNA.

12.2 An Origin Usually Initiates Bidirectional Replication A replication fork is initiated at the origin and then moves sequentially along DNA. Replication is unidirectional when a single replication fork is created at an origin. Replication is bidirectional when an origin creates two replication forks that move in opposite directions. Figure 12.02: Replicons may be unidirectional or bidirectional, depending on whether one or two replication forks are formed at the origin.

12.3 The Bacterial Genome Is (Usually) a Single Circular Replicon Bacterial replicons are usually circles that replicate bidirectionally from a single origin. The origin of E. coli, oriC, is 245 bp in length. The two replication forks usually meet halfway around the circle, but there are ter sites that cause termination if the replication forks go too far. Figure 12.04: Bidirectional replication of a circular bacterial chromosome is initiated at a single origin.

12.4 Methylation of the Bacterial Origin Regulates Initiation oriC also contains eleven GATC/CTAG repeats that are methylated on adenine on both strands. Replication generates hemimethylated DNA, which cannot initiate replication. Figure 12.05: The E. coli origin of replication, oriC contains multiple binding sites for the DnaA initiator protein. In a number of cases these sites overlap Dam methylation sites.

12.4 Methylation of the Bacterial Origin Regulates Initiation There is a 13-minute delay before the GATC/CTAG repeats are remethylated. SeqA binds to hemimethylated DNA and is required for delaying rereplication. Figure 12.06: Only fully methylated origins can initiate replication; hemimethylated daughter origins cannot be used again until they have been restored to the fully methylated state.

12.5 Initiation: Creating the Replication Forks at the Origin Initiation at oriC requires the sequential assembly of a large protein complex on the membrane. DnaA is the licensing factor (a factor necessary for replication; it is inactivated or destroyed after one round of replication). oriC must be fully methylated for replication to initiate. DnaA-ATP binds to short repeated sequences and forms an oligomeric complex that melts DNA.

12.5 Initiation: Creating the Replication Forks at the Origin Six DnaC monomers bind each hexamer of DnaB, and this complex binds the origin. A hexamer of DnaB forms the replication fork. Gyrase and SSB are also required. DnaG is bound to the helicase complex and creates the replication fork. Figure 12.07: The minimal origin is defined by the distance between the outside members of the 13-mer and 9-mer repeats.

12.5 Initiation: Creating the Replication Forks at the Origin Figure 12.08: Prepriming involves formation of a complex by sequential association of proteins, which leads to the separation of DNA strands.

12.6 Each Eukaryotic Chromosome Contains Many Replicons A eukaryotic chromosome is divided into many replicons. The progression into S phase is tightly controlled. Figure 12.09: A eukaryotic chromosome contains multiple origins of replication that ultimately merge during replication.

12.6 Each Eukaryotic Chromosome Contains Many Replicons Eukaryotic replicons are 40 to 100 kb in length. Individual replicons are activated at characteristic times during S phase. Regional activation patterns suggest that replicons near one another are activated at the same time.

12.7 Replication Origins Bind the ORC Origins in S. cerevisiae are short A-T sequences that have an essential 11 bp sequence. The ORC is a complex of six proteins that binds to an ARS (an origin for replication in yeast). The common feature among different examples of these sequences is a conserved 11 bp sequence called the A domain. Figure 12.10: An ARS extends for ~50 bp and includes a consensus sequence (A) and additional elements (B1–B3).

12.7 Replication Origins Bind the ORC Related ORC complexes are found in multicellular eukaryotes.

12.8 Licensing Factor Controls Eukaryotic Rereplication Licensing factor is necessary for initiation of replication at each origin. Licensing factor is present in the nucleus prior to replication, but is removed, inactivated, or destroyed by replication.

12.8 Licensing Factor Controls Eukaryotic Rereplication Initiation of another replication cycle becomes possible only after licensing factor re-enters the nucleus after mitosis. Figure 12.12: Licensing factor in the nucleus is inactivated after replication. A new supply of licensing factor can enter only when the nuclear membrane breaks down at mitosis.

12.8 Licensing Factor Controls Eukaryotic Rereplication The ORC is a protein complex that is associated with yeast origins throughout the cell cycle. Cdc6 protein is an unstable protein that is synthesized only in G1. Cdc6 binds to ORC and allows MCM proteins to bind. Figure 12.13: Proteins at the origin control susceptibility to initiation.

12.8 Licensing Factor Controls Eukaryotic Rereplication When replication is initiated, Cdc6, Cdt1, and MCM proteins are displaced. The degradation of Cdc6 prevents reinitiation. prereplication complex – A protein-DNA complex at the origin in S. cerevisiae that is required for DNA replication. The complex contains the ORC complex, Cdc6, and the MCM proteins. postreplication complex – A protein-DNA complex in S. cerevisiae that consists of the ORC complex bound to the origin.

12.8 Licensing Factor Controls Eukaryotic Rereplication Some MCM proteins are in the nucleus throughout the cell cycle, but others may enter only after mitosis.