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

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

3. 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.

4. - Usually a very long linear molecular of DNA
E.coli replication
Human replication
The origin of E. coli, oriC, 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.

5. 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.

6. Figure 12.B01: The Messelson-Stahl experiment showing that replication is semiconservative.

7. 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.

8. Figure 12.03: A replication bubble forms a θ structure in circular DNA.

9. 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.

10. When replication fork meet the protein-bound DNA
Repressor  replaced and rebind
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.

11. 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. 11 copies of palindromic GATC seq; methylated at N6 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, oriC contains multiple binding sites for the DnaA initiator protein. In a number of cases these sites overlap Dam methylation sites.

13. 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.

14. SeqA is not seq specific binding protein hemi-Me binding DnaA can displace it
SeqA binding  blocking Dam (delay re-methylation) DnaA binding blocking, DnaA expression suppression
seqA-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)

15. 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.

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

17. 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.

18. DnaA-ATP binding to 9mer repeated region
DnaA act on 13mer region (AT rich)
Open bubble formation
Transcription aid DNA torsion
DnaA recruites DnaB/DnaC complex (role of DnaC; chaperon); repriming stage
Six DnaC monomer binding to DnaB hexamer
DnaA is displaced
Hexemeric DnaB extend open region using helicase
Gyrase elimination of torsion
SSB
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.

20. 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.

21. 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: 2000bp/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

22. 12.7 Replication Origins Bind the ORC
Multi-origin in eukaryotic cell
ARS; autonomously replicating sequence
Mutation analysis 14bp AT rich core region (A domain)
Origins in S. cerevisiae are short A-T sequences that have an essential 11 bp sequence (ACS; ARS consensus sequence).
B elements
Figure 12.10: An ARS extends for ~50 bp and includes a consensus sequence (A) and additional elements (B1–B3).

23. The ORC is a complex of six proteins (-400KD) that binds to an ARS (an origin for replication in yeast).
ORC binds to A and B1 elements
Through ORC binding site, 400 origins are estimated (35Kb 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.
scORC, spORC, DmORC, XIORC

24. 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.

25. 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.

26. 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.
Regulation of cell division one by one cycle
Preventing unnecessary division

27. 12.8 Licensing Factor Controls Eukaryotic Rereplication
The ORC is a protein complex that is associated with yeast origins throughout the cell cycle.
A-B1 elements is protected by ORC from Dnase. However, center of B1 is hypersensitive to Dnase
Hypersensitivity is diminished during G1 process by cdc6
Cdc6 protein is an unstable protein that is synthesized only in G1 (half life 5 min).
In mammal, cdc6 is phosphorylated and exported from nucleus
Cdc6 binds to ORC and allows MCM proteins to bind.
Figure 12.13: Proteins at the origin control susceptibility to initiation.

28. MCM2, 3, 5 enter the nucleus during mitosis
Mammalian MCM3 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, 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.
Figure 12.13: Proteins at the origin control susceptibility to initiation.

30. 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
DnaA and ORC: more than DNA replication initiators
The bacterial DnaA and the eukaryotic origin recognition complex (ORC, which is composed of six subunits referred to as Orc1–Orc6) are functional homologues and share several features and activities (Figure I). First, all initiators contain specific motifs that facilitate DNA binding. While DnaA 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, DnaA 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 DnaA 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 N-terminal domain of DnaA interacts directly with the helicase to load the enzyme onto DNA, whereas in eukaryotes ORC recruits two additional proteins, Cdc6 and Ctd1, 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. DnaA can be inactivated through hydrolysis of its bound ATP, while ORC inactivation is more complex and is thought to involve ATP hydrolysis, loss of Cdc6/Cdt1 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

31. Figure 12.HP01A: The Meselson-Stahl Experiment

32. Figure 12.HP01B: The Meselson-Stahl Experiment