Chapter 12 The Replicon Initiation of Replication Jocelyn

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Chapter 12 The Replicon : Initiation of Replication Jocelyn E. Krebs

Chapter 12 The Replicon : Initiation of Replication Jocelyn E. Krebs

Figure 12. CO © Laguna Design/Photo Researchers, Inc.

Figure 12. CO © Laguna Design/Photo Researchers, Inc.

12. 1 Introduction • replicon – A unit of the genome in which DNA

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. • 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.

E. coli replication Human replication The origin of E. coli, ori. C, is 245

E. coli replication Human replication The origin of E. coli, ori. C, is 245 bp in length. Eukaryotic genome - Usually a very long linear molecular of DNA - Contains a large number of replicons spaced unevenly throughout the chromosome - Eukaryotic origin usage is controlled by the ability of regulator proteins to bind to it Prokaryotic genome -Usually a single circular molecular of DNA - Has a single replication origin (depends on a single origin initiation event that occurs at the unique origin - The frequency of initiation at the bacterial origin is controlled by its state of methylation.

12. 2 An Origin Usually Initiates Bidirectional Replication • semiconservative replication – Replication accomplished

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 Figure 12. 01: Replicated DNA is seen replication bubble within as a replication bubble flanked by nonreplicated DNA.

Figure 12. B 01: The Messelson-Stahl experiment showing that replication is semiconservative.

Figure 12. B 01: The Messelson-Stahl experiment showing that replication is semiconservative.

12. 2 An Origin Usually Initiates Bidirectional Replication • A replication fork is initiated

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.

Figure 12. 03: A replication bubble forms a θ structure in circular DNA.

Figure 12. 03: A replication bubble forms a θ structure in circular DNA.

12. 3 The Bacterial Genome Is (Usually) a Single Circular Replicon Figure 12. 04:

12. 3 The Bacterial Genome Is (Usually) a Single Circular Replicon Figure 12. 04: Bidirectional replication of a circular bacterial chromosome is initiated at a single origin. • Bacterial replicons are usually circles that replicate bidirectionally from a single origin. • The origin of E. coli, ori. C, 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.

When replication fork meet the protein-bound DNA 1. Repressor replaced and rebind 2. Transcription

When replication fork meet the protein-bound DNA 1. Repressor replaced and rebind 2. Transcription factor; replication fork is faster more 10 time than RNA pol. - The same direction; waiting for RNA pol - Opposite direction; stalled and transcription -coupled repair system displace RNA pol replication fork ongoing 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 • ori. C also contains

12. 4 Methylation of the Bacterial Origin Regulates Initiation • ori. C 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, ori. C contains multiple binding sites for the Dna. A initiator protein. In a number of cases these sites overlap Dam methylation sites.

11 copies of palindromic GATC seq; methylated at N 6 of adenine (Dam methylase)

11 copies of palindromic GATC seq; methylated at N 6 of adenine (Dam methylase) Both strands are methylated before replication hemimethylation Hemimethylated ori did not served as replication origin Unmethylated Ori can be used for replication origin hemi-Me inhibits replication Figure 12. 05: The E. coli origin of replication, ori. C contains multiple binding sites for the Dna. A initiator protein. In a number of cases these sites overlap Dam methylation sites.

 • There is a 13 -minute delay before the GATC/CTAG repeats are remethylated.

• There is a 13 -minute delay before the GATC/CTAG repeats are remethylated. • Seq. A 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.

 • Seq. A is not seq specific binding protein hemi-Me binding Dna. A

• Seq. A is not seq specific binding protein hemi-Me binding Dna. A can displace it • Seq. A binding blocking Dam (delay re-methylation) Dna. A binding blocking, Dna. A expression suppression • seq. A-mediated sequestering increase intermediate phase (cell cycle delay) • Cell membrane related event • Methylation also contribute to discern the new and old strands and repair system (recognize which one is template)

12. 5 Initiation: Creating the Replication Forks at the Origin • Initiation at ori.

12. 5 Initiation: Creating the Replication Forks at the Origin • Initiation at ori. C requires the sequential assembly of a large protein complex on the membrane. • Dna. A is the licensing factor (a factor necessary for replication; it is inactivated or destroyed after one round of replication). • ori. C must be fully methylated for replication to initiate. • Dna. A-ATP binds to short repeated sequences and forms an oligomeric complex that melts DNA.

Required event for replication initiation 1. Protein synthesis Dna. A (lisensing factor) 2. Transcription

Required event for replication initiation 1. Protein synthesis Dna. A (lisensing factor) 2. Transcription activation (replication origin structure) 3. Membrane/wall synthesis Initiation six proteins; Dna. A; unique protein of Ori. C, ATP binding protein, ATPase, activated by membrane phosphlipid and single strand DNA (* Dna. A –ATP is full active form) Dna. B: helicase (ATP hydrolysis-dependent 5 -3) engine fork process Dna. C: Dna. B binding protein, HU; DNA binding protein, Gyrase; toposiomerase II, SSB; single strand DNA bindign protein

12. 5 Initiation: Creating the Replication Forks at the Origin • Six Dna. C

12. 5 Initiation: Creating the Replication Forks at the Origin • Six Dna. C monomers bind each hexamer of Dna. B, and this complex binds the origin. • A hexamer of Dna. B forms the replication fork. Gyrase and SSB are also required. • Dna. G 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.

1. Dna. A-ATP binding to 9 mer repeated region 2. Dna. A act on

1. Dna. A-ATP binding to 9 mer repeated region 2. Dna. A act on 13 mer region (AT rich) 3. Open bubble formation 4. Transcription aid DNA torsion 5. Dna. A recruites Dna. B/Dna. C complex (role of Dna. C; chaperon); repriming stage 6. Six Dna. C monomer binding to Dna. B hexamer 7. Dna. A is displaced 8. Hexemeric Dna. B extend open region using helicase 9. Gyrase elimination of torsion 10. SSB 11. HU bend DNA ATP required (helicase and Gyrase) 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

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

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 (regional control). • Replication rate: 2000 bp/min (5000 bp/min in bacteria) • Because of chromatin • Hypothetically, 1 hr is enough for replication. But actually, 6 hr is required for S-phase only 15% origin is involved • Active gene-related origin • No termination region

12. 7 Replication Origins Bind the ORC • Multi-origin in eukaryotic cell • ARS;

12. 7 Replication Origins Bind the ORC • Multi-origin in eukaryotic cell • ARS; autonomously replicating sequence • Mutation analysis 14 bp AT rich core region (A domain) Figure 12. 10: An ARS extends for ~50 bp and includes a consensus sequence (A) and additional elements (B 1–B 3). • Origins in S. cerevisiae are short A-T sequences that have an essential 11 bp sequence (ACS; ARS consensus sequence). • B elements

 • The ORC is a complex of six proteins (-400 KD) that binds

• The ORC is a complex of six proteins (-400 KD) that binds to an ARS (an origin for replication in yeast). • ORC binds to A and B 1 elements • Through ORC binding site, 400 origins are estimated (35 Kb per ORC) • The common feature among different examples of these sequences is a conserved 11 bp sequence called the A domain. • Related ORC complexes are found in multicellular eukaryotes. • sc. ORC, sp. ORC, Dm. ORC, XIORC

12. 8 Licensing Factor Controls Eukaryotic Rereplication • Licensing factor is necessary for initiation

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.

In xenopus egg, without de novo synthesis, 11 divisions can be takes Cytoplasmic factors

In xenopus egg, without de novo synthesis, 11 divisions can be takes Cytoplasmic factors are required for continuous cell division Figure 12. 11: A nucleus injected into a Xenopus egg can replicate only once unless the nuclear membrane is permeabilized to allow subsequent replication cycles.

12. 8 Licensing Factor Controls Eukaryotic Rereplication • Initiation of another replication cycle becomes

12. 8 Licensing Factor Controls Eukaryotic Rereplication • Initiation of another replication cycle becomes possible only after licensing factor re-enters the nucleus after mitosis. Regulation of cell division one by one cycle Preventing unnecessary division 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 • • • Figure 12. 13: Proteins

12. 8 Licensing Factor Controls Eukaryotic Rereplication • • • Figure 12. 13: Proteins at the origin control susceptibility to initiation. The ORC is a protein complex that is associated with yeast origins throughout the cell cycle. A-B 1 elements is protected by ORC from Dnase. However, center of B 1 is hypersensitive to Dnase Hypersensitivity is diminished during G 1 process by cdc 6 Cdc 6 protein is an unstable protein that is synthesized only in G 1 (half life 5 min). In mammal, cdc 6 is phosphorylated and exported from nucleus Cdc 6 binds to ORC and allows MCM proteins to bind.

MCM 2, 3, 5 enter the nucleus during mitosis Mammalian MCM 3 bind to

MCM 2, 3, 5 enter the nucleus during mitosis Mammalian MCM 3 bind to chromosome and release after replication But MCMs are retained in nucleus in animal cell, indicating that MCM is not one of licensing factor • • • When replication is initiated, Cdc 6, Cdt 1, and MCM proteins are displaced. The degradation of Cdc 6 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, Cdc 6, and the MCM proteins. postreplication complex – A protein-DNA complex in S. cerevisiae that consists of the ORC complex bound to the origin. Figure 12. 13: Proteins at the origin control susceptibility to initiation.

Trends in Cell Biology Volume 21, Issue 3 2011 188 - 194 Figure I

Trends in Cell Biology Volume 21, Issue 3 2011 188 - 194 Figure I Initiation of replication in bacteria and eukaryotes. Graham Scholefield , Jan-Willem Veening , Heath Murray Dna. A and ORC: more than DNA replication initiators The bacterial Dna. A and the eukaryotic origin recognition complex (ORC, which is composed of six subunits referred to as Orc 1–Orc 6) are functional homologues and share several features and activities (Figure I). First, all initiators contain specific motifs that facilitate DNA binding. While Dna. A recognizes specific DNA sequences within identified replication origins using its C-terminal helix-turn-helix (HTH) motif, the localization of ORC at replication origins in higher organisms appears to be less dependent on sequence and is thought to be mediated through potential DNA-binding motifs found within multiple ORC subunits. Second, Dna. A and ORC are ATP-binding proteins that contain homologous AAA+ motifs (present in five of the six Orc proteins). Functional and structural studies indicate that both Dna. A and ORC undergo ATP-dependent conformational changes that activate the proteins to allow the initiation of DNA replication. Third, initiator proteins assembled at replication origins load the replicative helicases required for bidirectional DNA replication elongation. In bacteria, the Nterminal domain of Dna. A interacts directly with the helicase to load the enzyme onto DNA, whereas in eukaryotes ORC recruits two additional proteins, Cdc 6 and Ctd 1, that are required for helicase loading. Lastly, both initiators are inactivated following DNA replication initiation to ensure that genome duplication occurs only once per cell cycle. Dna. A can be inactivated through hydrolysis of its bound ATP, while ORC inactivation is more complex and is thought to involve ATP hydrolysis, loss of Cdc 6/Cdt 1 binding and/or phosphorylation. Please see 48, 55, 56 and 57 for excellent reviews with more detailed information regarding the regulation and mechanisms of DNA replication initiator proteins in DNA synthesis

Figure 12. HP 01 A: The Meselson-Stahl Experiment

Figure 12. HP 01 A: The Meselson-Stahl Experiment

Figure 12. HP 01 B: The Meselson-Stahl Experiment

Figure 12. HP 01 B: The Meselson-Stahl Experiment