Replication Initiation and Termination in *E. coli*

F. Control Initiation at oriC at Fixed Growth Rate

F.1. Cell Cycle Regulation for oriC

• In organisms like E. coli, which divides approximately every $60$ minutes, the initiation of DNA replication at oriC occurs at a consistent rate.

• Each initiation event at oriC is precisely timed to match the cell's doubling time ($60$ minutes).

• This process is regulated by DnaA proteins.

• Roughly $11$ DnaA proteins typically bind to the oriC region.

• An additional approximately $200$ DnaA proteins exist in the cell but are not bound to oriC.

F.2. DnaA Protein Inactivation at oriC (RIDA)

• After replication initiation, DnaA protein bound at oriC is inactivated by a process called RIDA (Regulation Inactivation of DnaA).

• RIDA Mechanism:

1. DnaA(ATP) to DnaA(ADP) Conversion:

   • The active form, DnaA(ATP), interacts with HdaA (Homology to DnaA) protein within E. coli.

   • HdaA facilitates the hydrolysis of ATP bound to DnaA, converting DnaA(ATP) into DnaA(ADP) (the inactive form).

2. Release from oriC (Excluding High-Affinity Sites):

   • The now inactive DnaA(ADP) proteins are released from oriC.

   • Specifically, they are released from $8$ low-affinity double-stranded DNA (ds-DNA) sites and $3$ low-affinity single-stranded DNA (ss-DNA) sites.

   • Note that DnaA(ADP) is NOT released from the high-affinity ds-DNA sites designated R1, R2, and R4 within oriC.

3. Inactivation of oriC: If not all $11$ DnaA proteins are bound to oriC (implying the release of DnaA(ADP) from most sites), oriC becomes inactive for subsequent initiation events.

F.2.d. Inactivation of Unbound DnaA(ATP)

• The approximately $200$ DnaA(ATP) proteins not bound to oriC must also be inactivated to prevent unwanted binding to oriC and premature re-initiation.

• This inactivation occurs when DnaA(ATP) binds to a specific genomic locus called datA.

• datA Location and Function:

1. datA is situated at $95'$ on the E. coli genome, whereas oriC is at $83'$.

2. As the clockwise replication fork progresses from oriC, it reaches datA in approximately $8$ minutes.

3. Upon reaching datA, the cell now possesses $2$ copies of the datA site.

4. The presence of $2$ datA copies triggers the ATP hydrolysis of active DnaA(ATP), converting it into inactive DnaA(ADP).

• Aside: The question of why DnaA(ATP) doesn't bind to oriC during these $8$ minutes until datA becomes duplicated is addressed in section F.4.

F.3. "Recharging" DnaA(ADP) to DnaA(ATP)

• This process involves converting inactive DnaA(ADP) back to active DnaA(ATP) by replacing ADP with ATP.

• This "recharging" occurs later in the cell cycle.

• a. For DnaA(ADP) bound in oriC (at R1, R2, R4 sites):

• Recharging takes place when oriC is inserted into the cell membrane (as detailed in F.4.).

• b. For DnaA(ADP) NOT bound in oriC:

• Recharging happens when DnaA(ADP) binds to specific sites on the E. coli chromosome known as DARS1 and DARS2 (DnaA Reactivation Sequence).

• c. Summary Diagram:

• DnaA bound oriC $<br>ightarrow$ Inactivation by RIDA.

• DnaA NOT bound oriC $<br>ightarrow$ Inactivation by datA.

• Activation (recharging) occurs when oriC inserted in membrane or via DARS sites.

• ### F.4. oriC Regulation by Hemimethylated "Dam" Sites

  • a. Dam Sites in oriC:

  • oriC contains $11$ GATC motifs (Dam sites) within its $245$ bp sequence.

  • The Dam protein (DNA adenine methylase) methylates the adenine within the $5'-GATC-3'$ sequence, converting it to $6$-methyladenine.

  • After replication, the newly synthesized strand of DNA is unmethylated, resulting in a "hemimethylated" Dam site (one strand methylated, one unmethylated).

  • b. SeqA Protein Binding:

  1. Immediately after replication, the $11$ hemimethylated Dam sites within oriC are bound by the SeqA protein.

  2. SeqA (Sequestration protein A, a $21$ kD dimer) specifically binds only to hemimethylated Dam sites.

  3. Critically, SeqA only binds effectively when Dam sites are clustered at high density, a characteristic feature of oriC.

  • c. Membrane Insertion and Sequestration:

  1. Once SeqA binds, oriC is physically inserted into the cell membrane, typically at the mid-cell where active replication occurs.

  2. This membrane insertion prevents active DnaA(ATP) from binding to oriC, thereby inhibiting re-initiation.

  3. SeqA holds oriC in the membrane for approximately $15$ minutes, a duration sufficient for the datA site to be duplicated (which takes about $8$ minutes) and inactivate unbound DnaA(ATP).

  4. Multiple other clusters of high-density Dam sites (similar to oriC) located in a region of about $100,000$ bp between $83'$ and $85'$ on the chromosome are also inserted into the membrane for roughly $15$ minutes.

  • d. Impact on dnaA Gene Transcription and DnaA Levels:

  1. The dnaA gene itself is located at $83'$.

  2. Since the dnaA gene is also inserted into the membrane for this $15$-minute period, its transcription is suppressed.

  3. Consequently, dnaA mRNA is degraded, leading to a decline in the overall cellular concentration of DnaA protein.

  • e. Consequence: The combined effects of oriC sequestration in the membrane and reduced DnaA protein levels effectively prevent premature oriC re-initiation.

  • f. SeqA Release and New Initiation:

  1. After approximately $15$ minutes, SeqA releases oriC and the dnaA gene from the membrane.

  2. The dnaA gene can then be transcribed, leading to an increase in dnaA mRNA and DnaA protein levels.

  3. Once the DnaA concentration reaches a critical threshold, DnaA(ATP) binds to oriC, displaces the inhibitory Fis protein (which is present in oriC), and triggers a new round of DNA replication initiation.

G. Control of oriC Initiation with Changing Growth Conditions

1. Introduction

• E. coli exhibits remarkable flexibility in its division time, ranging from as fast as every $20$ minutes under optimal conditions to very slow rates under challenging conditions.

• oriC initiation is regulated differently to accommodate these varying growth conditions.

2. Regulation of oriC Activity

• oriC activity is modulated by the competitive balance between DnaA and Fis proteins.

• Active oriC (Increased Initiation): Occurs when DnaA levels are high and Fis levels are low.

• Inactive oriC (Slower Initiation): Occurs when DnaA levels are low and Fis levels are high.

• The cellular levels of DnaA and Fis proteins are primarily regulated at the level of their transcription.

• ### 3. Conditions Leading to Slower oriC Initiation

  • Slower oriC initiation (characterized by low DnaA and high Fis) is observed under conditions of nutrient limitation:

  • Low carbon and/or nitrogen availability.

  • Low phosphorus, low iron (Fe), or low fatty acid concentrations.

  • Low glucose levels.

  • (The specific molecular mechanisms by which these conditions regulate DnaA and Fis transcription were not provided).

H. Termination of Replication

1. Normal Replication Process

• DNA replication initiates at oriC.

• Two replication forks move bi-directionally (one clockwise, one counter-clockwise) around the circular chromosome.

• Termination typically occurs in a region opposite to oriC, near the end of the replication process.

2. Normal Termination Events

• The two replication forks meet in the termination (ter) region.

• Approximately $100$ bp of DNA remain unsynthesized by DNA polymerase III (DNAP III) at the meeting point.

• DNA polymerase I (DNAP I) then fills in these remaining gaps.

• DNA ligase seals any remaining single-strand breaks (nicks) in the phosphodiester backbone.

• The resulting two daughter chromosomes are often interlinked, forming structures called "catenated" DNA.

• A topoisomerase (specifically a type II topoisomerase, such as Topoisomerase IV in E. coli) is required to resolve these catenated DNA molecules, allowing the two circular chromosomes to separate.

• ### 3. "Replication Fork Traps"

  • Replication fork traps ensure that replication terminates within a specific region, even if the two forks travel at different speeds.

  • a. Asymmetrical Fork Stalling:

  • If the clockwise fork moves faster, it will be halted at terC, located at $33'$.

  • If the counter-clockwise fork moves faster, it will be halted at terA, located at $28'$.

  • b. ter Site Consensus Sequence:

  • terA, terC, and other ter sites (e.g., terB, terD, terE) share a consensus DNA sequence:
    $5'-AGTATGTTGTAACTAA-3'$

  • c. Mechanism of Trapping:

  • The Tus protein binds to these ter sites.

  • The Tus-ter complex acts as a polar barrier to the DNA helicase component of the replication machinery.

  • It has a "permissive side" where the DNA helicase is NOT blocked, allowing a fork to pass through.

  • It also has a "non-permissive side" where the DNA helicase IS blocked, arresting the fork.

  • This polarity ensures that forks can enter the termination region but are prevented from crossing a specific boundary, thereby localizing termination.