Replication Control in Prokaryotes and Eukaryotes

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Your Name

Published

February 5, 2025

Introduction

In the previous lecture, we examined the architecture of replication origins, including replicators and initiators. This lecture will now focus on the mechanisms by which prokaryotes and eukaryotes control DNA replication, highlighting both conserved and divergent strategies. Precise control of replication is essential to ensure complete genome duplication exactly once per cell cycle. Failure to achieve this can lead to unequal chromosome segregation and loss of genetic information. We will begin by discussing the initiation of replication in Escherichia coli (E. coli).

Replication Control in Prokaryotes

Initiation of Replication in Escherichia coli

Replication in E. coli initiates at a unique origin, OriC, which acts as a single replicon. The process is tightly regulated to ensure only one round of replication per cell cycle.

Replicator (`OriC`) and Initiator (DNA A)

Definition 1. The OriC locus contains specific DNA sequences that function as the replicator. Initiation begins with the binding of the initiator protein, DNA A, to nonamer repeat sequences within OriC. DNA A is an ATP-dependent protein; ATP binding is required for its activity.

Origin Denaturation

DNA A binding facilitates the denaturation of adjacent AT-rich 13-mer repeats in OriC. The lower stability of AT base pairs allows for easier strand separation. This denaturation, requiring ATP hydrolysis by DNA A, creates an opening in the DNA duplex, providing access for subsequent replication proteins.

Helicase Loading

The single-stranded DNA region created by DNA A allows for the loading of the replicative helicase, DNA B (hexamer), by its loader protein, DNA C. Once loaded, DNA B encircles the DNA and DNA C is released.

Primer Synthesis and Replisome Assembly

After helicase loading and further unwinding, primase (an RNA polymerase) synthesizes a short RNA primer on each template strand. These primers provide the 3’-OH ends necessary for DNA Polymerase III to begin DNA synthesis. Subsequently, DNA A detaches, and the DNA Polymerase III holoenzyme, the primary replication machinery in E. coli, is assembled at the primed origins.

Bidirectional Replication

Replication proceeds bidirectionally from OriC, forming two replication forks that move in opposite directions. DNA Polymerase III synthesizes both leading and lagging strands in a semi-discontinuous manner.

Regulation of Replication Re-initiation

To prevent multiple rounds of replication within a single cell cycle, E. coli employs a mechanism involving GATC sequences, Dam methyltransferase, and SeqA protein.

`GATC` Methylation and Hemimethylation

OriC is rich in GATC palindromic sequences, which are targets for Dam methyltransferase. Full methylation (dimethylation) of adenine residues in GATC on both strands is required for replication initiation. Immediately after replication, GATC sites become hemimethylated because Dam methyltransferase methylates newly synthesized strands more slowly.

SeqA Inhibition of Re-initiation

Hemimethylated GATC sequences are specifically bound by SeqA protein. SeqA binding inhibits re-initiation by:

Blocking DNA A access: SeqA prevents DNA A from binding to nonamer repeats, thus inhibiting new initiation events.
Inhibiting Dam methyltransferase: SeqA inhibits Dam methyltransferase activity at hemimethylated sites, prolonging the hemimethylated state.

SeqA binding is transient. Over time, SeqA dissociates, allowing Dam methyltransferase to fully methylate GATC sites. Full methylation is required for subsequent rounds of replication initiation, ensuring replication occurs only once per cell cycle.

Coordination of Replication and Cell Division

Temporal Discrepancy and Overlapping Cycles

The E. coli genome replication time (approximately 40 minutes) exceeds its cell division time (approximately 20 minutes). To accommodate this, replication and cell division cycles overlap.

Partial Diploidy in Daughter Cells

New replication initiation occurs before cell division is complete. Consequently, daughter cells inherit partially replicated chromosomes, resulting in partial diploidy. Regions near OriC are present in two copies due to replication initiation for the next generation, while regions further from OriC remain haploid until replicated. This contrasts with eukaryotes, where chromosome segregation is strictly coupled to complete genome replication.

Replication Control in Eukaryotes

Eukaryotic replication control is more complex than in prokaryotes, primarily due to the larger genome size and the presence of multiple chromosomes. Eukaryotic cells utilize multiple origins of replication and a tightly regulated, two-step mechanism to ensure replication occurs once and only once per cell cycle.

Eukaryotic Origins of Replication

Eukaryotic origins of replication share a similar architecture with prokaryotic origins but are more numerous and less defined by specific consensus sequences, except in yeast.

Homologues of Prokaryotic Replicator Elements

Definition 2 (Origin Recognition Elements ()). These sequences are functionally homologous to the nonamer sequences in E. coli* OriCand serve as binding sites for initiator proteins.*

Definition 3 (DNA Unwinding Elements ()). Functionally homologous to the 13-mer sequences in E. coli* OriC, are AT-rich regions that facilitate initial DNA denaturation.*

in Yeast: A Model System

Definition 4 (Autonomously Replicating Sequences () in Yeast). Autonomously Replicating Sequences () in yeast (Saccharomyces cerevisiae) are the best-characterized eukaryotic origins. are short (100-200 bp), AT-rich sequences containing ORE-like and DUE-like elements. They serve as a valuable model for understanding eukaryotic replication initiation due to their defined structure and the genetic tools available in yeast.

Temporal Control of Replication Origin Firing

Eukaryotic genomes are replicated from multiple origins that are activated at different times during S phase, following a temporal program.

Multiple Replication Origins and Replicons

Eukaryotes utilize multiple origins of replication distributed across each chromosome to ensure timely replication of their large genomes. These origins define multiple replicons, spaced approximately 100 kb apart on average.

Replication Timing Program: Early and Late Origins

Replication origins in eukaryotes do not fire simultaneously. A temporal program dictates that some origins fire early in S phase (early origins), while others fire later (late origins). This temporal order is linked to chromatin structure and transcriptional activity.

Replication Foci and Chromatin Domains

Replication origins cluster into replication foci within the nucleus.

Early Replication Foci: These foci, enriched in early firing origins, are typically located in the nucleoplasm and associated with euchromatin—less condensed, transcriptionally active chromatin.
Late Replication Foci: These foci, containing late firing origins, are often found near the nuclear periphery and nucleolus, associated with heterochromatin—more condensed, transcriptionally inactive chromatin, including centromeres and telomeres.

The later replication of heterochromatin may be related to its condensed structure and the need to protect it from potential damage during replication.

Experimental Mapping of Replication Timing

Replication timing can be experimentally mapped using nucleotide analogues like bromodeoxyuridine (BrdU). BrdU is incorporated into newly synthesized DNA and can be detected with specific antibodies. By analyzing BrdU incorporation at different time points during S phase, researchers can identify early and late replicating regions of the genome, confirming the temporal program of origin firing and its correlation with chromatin domains.

Regulation of Replication Initiation: The Pre-Replicative Complex (pre-RC)

Eukaryotic cells employ a two-step mechanism to ensure that each replication origin is activated only once per cell cycle. This involves the assembly of an inactive pre-replicative complex (pre-RC) during G1 phase (licensing) and its activation in S phase (origin firing).

pre-RC Assembly in G1 Phase: Licensing Origins

In G1 phase, when Cyclin-Dependent Kinase (CDK1) activity is low, pre-replicative complexes () are assembled at replication origins. This process "licenses" origins for replication, ensuring they are competent to initiate replication in the subsequent S phase but prevents premature firing.

pre-RC Components: ORC, MCM2-7, CDC6, and CDT1

Definition 5 (Pre-Replicative Complex (pre-RC)). In G1 phase, when Cyclin-Dependent Kinase (CDK1) activity is low, pre-replicative complexes () are assembled at replication origins. This complex licenses origins for replication.

Definition 6 (Origin Recognition Complex (ORC)). The eukaryotic initiator protein, homologous to DNA A in prokaryotes. ORC binds to throughout the cell cycle, serving as a platform for pre-RC assembly.

Definition 7 (MCM2-7 Complex (MCM2-7)). The replicative helicase in eukaryotes, homologous to DNA B. The inactive MCM2-7 complex is loaded onto origins during pre-RC formation.

Definition 8 (CDC6 and CDT1). Helicase loaders, homologous to DNA C. CDC6 and CDT1 are essential for loading MCM2-7 onto ORC at origins during G1 phase.

Helicase Inactivation within the pre-RC

While CDC6 and CDT1 facilitate MCM2-7 loading, they also maintain MCM2-7 in an inactive state within the pre-RC in G1. These loaders effectively clamp the helicase onto DNA, preventing premature DNA unwinding.

Activation of Origins at the G1/S Transition

The transition from G1 to S phase triggers pre-RC activation and origin firing. This activation is driven by the increased activity of Cyclin-Dependent Kinases (CDK1, specifically CDK11) and Dbf4-dependent kinase (DDK).

Role of CDK1 and DDK in Origin Firing

Definition 9 (Cyclin-Dependent Kinase 1 (CDK1)). CDK1 activity increases at the G1/S transition and phosphorylates CDC6 and CDT1.

Definition 10 (Dbf4-dependent kinase (DDK)). DDK is activated in early S phase and phosphorylates MCM2-7 helicases and other pre-RC components.

Phosphorylation-Dependent Loader Degradation and Helicase Activation

Phosphorylation of CDC6 and CDT1 by CDK1 leads to:

Loader Degradation: Phosphorylation triggers the degradation of CDC6 and CDT1, preventing further MCM2-7 loading and re-replication at already fired origins.
Helicase Activation: Phosphorylation events, including MCM2-7 phosphorylation by DDK, activate the MCM2-7 helicase complex.

Origin Unwinding and Replication Fork Formation

Activated MCM2-7 helicases initiate DNA unwinding at the origin, creating a replication bubble. This allows for the recruitment of other replication factors and the establishment of active replication forks.

Sequential Recruitment of Replication Enzymes

Following origin unwinding, replication enzymes are recruited in a specific order. Primase is recruited first to synthesize RNA primers. Subsequently, processive DNA polymerases are recruited to initiate DNA synthesis. Note: The transcript mentions processive polymerases entering before primase, which is likely an error as primase is required to synthesize the RNA primer for DNA polymerase to initiate extension.

Coordination by PCNA and RFC

Proliferating Cell Nuclear Antigen (PCNA), the sliding clamp, and Replication Factor C (RFC), the clamp loader, are crucial for eukaryotic replication. PCNA enhances DNA polymerase processivity and, along with RFC, coordinates leading and lagging strand synthesis and chromatin reassembly.

Epigenetic Inheritance during Replication

Definition 11 (Epigenetic Inheritance). Epigenetic inheritance is the process by which epigenetic information, heritable changes in gene expression without alterations to the DNA sequence, is transmitted across cell divisions.

Epigenetic Stability and Cellular Identity

Importance of Epigenetic Stability

Definition 12 (Epigenetic Stability). Epigenetic stability is essential for preserving tissue identity and function.

Epigenetic Marks and Tissue-Specific Functions

Definition 13 (Epigenetic Marks). Tissue-specific functions are dictated by distinct patterns of gene expression, which are established and maintained by epigenetic marks, including histone modifications and DNA methylation.

Epigenetic Plasticity vs. Stability

Definition 14 (Epigenetic Plasticity). Epigenetic plasticity is the ability to dynamically alter epigenetic marks in response to environmental signals, is also important for development and adaptation.

Mechanisms of Histone Modification Inheritance

Histone modifications are key epigenetic marks. Their inheritance during DNA replication ensures the propagation of chromatin states and gene expression patterns.

Semi-Conservative Distribution of Parental Histones

Definition 15 (Semi-Conservative Distribution of Parental Histones). During DNA replication, parental histones are distributed to daughter DNA strands. Specifically, H3-H4 tetramers are distributed semi-conservatively, meaning they are roughly equally segregated to both daughter DNA molecules.

Differential Segregation of Histone Subunits

H3-H4 tetramers and H2A-H2B dimers exhibit different segregation patterns. H3-H4 tetramers tend to remain associated with DNA and are inherited, while H2A-H2B dimers are moreExchangeable and are replaced with both parental and newly synthesized dimers. This distinction is significant as H3 and H4 carry a substantial portion of pre-existing histone modifications.

Reader-Writer Complexes for Modification Re-establishment

Definition 16 (Reader-Writer Complexes). Reader-writer complexes function by reading specific histone modifications on parental histones and writing the same modifications on newly deposited histones in proximity.

Reader Domains: Bromodomains and Chromodomains

Definition 17 (Reader Domains). Reader domains are protein modules that specifically bind to histone modifications.

Definition 18 (Bromodomains). Bromodomains recognize and bind to acetylated lysine residues, typically associated with transcriptional activation.

Definition 19 (Chromodomains). Chromodomains recognize and bind to methylated lysine residues, and in some cases, deacetylated lysine, involved in both transcriptional activation and repression depending on context.

Writer Domains: and

Definition 20 (Writer Domains). Writer domains are enzymatic domains that catalyze the addition or removal of histone modifications.

Definition 21 (Histone Acetyltransferases ()). Histone Acetyltransferases () add acetyl groups to lysine residues, generally associated with transcriptional activation.

Definition 22 (Histone Deacetylases ()). Histone Deacetylases () remove acetyl groups, typically associated with transcriptional repression.

Histone Chaperones in Nucleosome Assembly and PCNA Coordination

Definition 23 (Histone Chaperones). Histone chaperones are essential for nucleosome assembly during replication.

Definition 24 (Nucleosome Assembly Protein 1 (NAP1)). Nucleosome Assembly Protein 1 (NAP1) facilitates the deposition of H2A-H2B dimers onto DNA.

Definition 25 (Chromatin Assembly Factor 1 (CAF1)). Chromatin Assembly Factor 1 (CAF1) specifically deposits H3-H4 tetramers onto newly replicated DNA. CAF1 is recruited to replication forks by interacting with PCNA.

Histone H1, Nucleophosmin, and Chromatin Compaction

Definition 26 (Histone H1). Histone H1, a linker histone, is crucial for higher-order chromatin compaction (transition from 10nm to 30nm fiber).

Definition 27 (Nucleophosmin). Nucleophosmin is a chaperone specifically for histone H1, facilitating its incorporation into chromatin.

Clinical Relevance: Nucleophosmin in Leukemia

Nucleophosmin is an abundant nucleolar and nucleoplasmic protein involved in ribosome biogenesis and histone H1 chaperoning. Mutations in the NPM1 gene, encoding nucleophosmin, are frequent in acute myeloid leukemia (AML), accounting for approximately 30% of cases. These mutations often lead to aberrant cytoplasmic localization of nucleophosmin and contribute to leukemogenesis, highlighting the functional importance of proper chromatin assembly and epigenetic regulation in preventing disease.

Conclusion

This lecture has detailed the mechanisms controlling DNA replication in prokaryotes and eukaryotes, emphasizing the critical role of precise regulation for genome stability and the inheritance of both genetic and epigenetic information. Eukaryotic replication is characterized by multiple origins, temporal control, and a sophisticated licensing system involving the pre-RC to ensure once-per-cycle replication. Furthermore, epigenetic inheritance, particularly the transmission of histone modifications through reader-writer complexes and histone chaperones coordinated with replication, is essential for maintaining cellular identity and tissue homeostasis.

Key Takeaways:

Prokaryotic replication at OriC is regulated by DNA A, ATP hydrolysis, and GATC methylation/hemimethylation cycle involving SeqA to prevent re-initiation.
Eukaryotic replication features multiple origins with a temporal firing program linked to chromatin structure and is licensed by pre-RC assembly in G1 and activation by CDK1 and DDK in S phase.
Epigenetic inheritance, especially histone modification inheritance, is crucial for maintaining cell identity and tissue homeostasis.
Reader-writer complexes and histone chaperones like CAF1 and NAP1, coordinated by PCNA, are essential for propagating epigenetic information during replication.

Further Questions and Next Lecture Topics: The next lecture will address the end-replication problem of linear chromosomes and the role of telomeres and telomerase, along with their implications in aging and cancer. We will also further explore the pathological consequences of dysregulation in replication control and epigenetic inheritance in diseases such as leukemia.

--- title: "Replication Control in Prokaryotes and Eukaryotes" author: "Your Name" date: "2025-02-05" format: html: toc: true # Table of Contents toc-depth: 2 code-tools: true theme: cosmo # Or "journal" for Distill-like minimalism --- # Introduction In the previous lecture, we examined the architecture of replication origins, including replicators and initiators. This lecture will now focus on the mechanisms by which prokaryotes and eukaryotes control DNA replication, highlighting both conserved and divergent strategies. Precise control of replication is essential to ensure complete genome duplication exactly once per cell cycle. Failure to achieve this can lead to unequal chromosome segregation and loss of genetic information. We will begin by discussing the initiation of replication in *Escherichia coli* (*E. coli*). # Replication Control in Prokaryotes ## Initiation of Replication in *Escherichia coli* Replication in *E. coli* initiates at a unique origin, `OriC`, which acts as a single replicon. The process is tightly regulated to ensure only one round of replication per cell cycle. ### Replicator (`OriC`) and Initiator (DNA A) ::: definition **Definition 1**. *The `OriC` locus contains specific DNA sequences that function as the replicator. Initiation begins with the binding of the initiator protein, DNA A, to nonamer repeat sequences within `OriC`. DNA A is an ATP-dependent protein; ATP binding is required for its activity.* ::: ### Origin Denaturation DNA A binding facilitates the denaturation of adjacent AT-rich 13-mer repeats in `OriC`. The lower stability of AT base pairs allows for easier strand separation. This denaturation, requiring ATP hydrolysis by DNA A, creates an opening in the DNA duplex, providing access for subsequent replication proteins. ### Helicase Loading The single-stranded DNA region created by DNA A allows for the loading of the replicative helicase, DNA B (hexamer), by its loader protein, DNA C. Once loaded, DNA B encircles the DNA and DNA C is released. ### Primer Synthesis and Replisome Assembly After helicase loading and further unwinding, primase (an RNA polymerase) synthesizes a short RNA primer on each template strand. These primers provide the 3'-OH ends necessary for DNA Polymerase III to begin DNA synthesis. Subsequently, DNA A detaches, and the DNA Polymerase III holoenzyme, the primary replication machinery in *E. coli*, is assembled at the primed origins. ### Bidirectional Replication Replication proceeds bidirectionally from `OriC`, forming two replication forks that move in opposite directions. DNA Polymerase III synthesizes both leading and lagging strands in a semi-discontinuous manner. ## Regulation of Replication Re-initiation To prevent multiple rounds of replication within a single cell cycle, *E. coli* employs a mechanism involving `GATC` sequences, Dam methyltransferase, and SeqA protein. ### `GATC` Methylation and Hemimethylation `OriC` is rich in `GATC` palindromic sequences, which are targets for Dam methyltransferase. Full methylation (dimethylation) of adenine residues in `GATC` on both strands is required for replication initiation. Immediately after replication, `GATC` sites become hemimethylated because Dam methyltransferase methylates newly synthesized strands more slowly. ### SeqA Inhibition of Re-initiation Hemimethylated `GATC` sequences are specifically bound by SeqA protein. SeqA binding inhibits re-initiation by: 1. **Blocking DNA A access:** SeqA prevents DNA A from binding to nonamer repeats, thus inhibiting new initiation events. 2. **Inhibiting Dam methyltransferase:** SeqA inhibits Dam methyltransferase activity at hemimethylated sites, prolonging the hemimethylated state. SeqA binding is transient. Over time, SeqA dissociates, allowing Dam methyltransferase to fully methylate `GATC` sites. Full methylation is required for subsequent rounds of replication initiation, ensuring replication occurs only once per cell cycle. ## Coordination of Replication and Cell Division ### Temporal Discrepancy and Overlapping Cycles The *E. coli* genome replication time (approximately 40 minutes) exceeds its cell division time (approximately 20 minutes). To accommodate this, replication and cell division cycles overlap. ### Partial Diploidy in Daughter Cells New replication initiation occurs before cell division is complete. Consequently, daughter cells inherit partially replicated chromosomes, resulting in partial diploidy. Regions near `OriC` are present in two copies due to replication initiation for the next generation, while regions further from `OriC` remain haploid until replicated. This contrasts with eukaryotes, where chromosome segregation is strictly coupled to complete genome replication. # Replication Control in Eukaryotes Eukaryotic replication control is more complex than in prokaryotes, primarily due to the larger genome size and the presence of multiple chromosomes. Eukaryotic cells utilize multiple origins of replication and a tightly regulated, two-step mechanism to ensure replication occurs once and only once per cell cycle. ## Eukaryotic Origins of Replication Eukaryotic origins of replication share a similar architecture with prokaryotic origins but are more numerous and less defined by specific consensus sequences, except in yeast. ### Homologues of Prokaryotic Replicator Elements ::: definition **Definition 2** (Origin Recognition Elements ()). *These sequences are functionally homologous to the nonamer sequences in *E. coli* `OriC`and serve as binding sites for initiator proteins.* ::: ::: definition **Definition 3** (DNA Unwinding Elements ()). *Functionally homologous to the 13-mer sequences in *E. coli* `OriC`, are AT-rich regions that facilitate initial DNA denaturation.* ::: ### in Yeast: A Model System ::: definition **Definition 4** (Autonomously Replicating Sequences () in Yeast). *Autonomously Replicating Sequences () in yeast (*Saccharomyces cerevisiae*) are the best-characterized eukaryotic origins. are short (100-200 bp), AT-rich sequences containing ORE-like and DUE-like elements. They serve as a valuable model for understanding eukaryotic replication initiation due to their defined structure and the genetic tools available in yeast.* ::: ## Temporal Control of Replication Origin Firing Eukaryotic genomes are replicated from multiple origins that are activated at different times during S phase, following a temporal program. ### Multiple Replication Origins and Replicons Eukaryotes utilize multiple origins of replication distributed across each chromosome to ensure timely replication of their large genomes. These origins define multiple replicons, spaced approximately 100 kb apart on average. ### Replication Timing Program: Early and Late Origins Replication origins in eukaryotes do not fire simultaneously. A temporal program dictates that some origins fire early in S phase (early origins), while others fire later (late origins). This temporal order is linked to chromatin structure and transcriptional activity. ### Replication Foci and Chromatin Domains Replication origins cluster into replication foci within the nucleus. - **Early Replication Foci:** These foci, enriched in early firing origins, are typically located in the nucleoplasm and associated with euchromatin---less condensed, transcriptionally active chromatin. - **Late Replication Foci:** These foci, containing late firing origins, are often found near the nuclear periphery and nucleolus, associated with heterochromatin---more condensed, transcriptionally inactive chromatin, including centromeres and telomeres. The later replication of heterochromatin may be related to its condensed structure and the need to protect it from potential damage during replication. ### Experimental Mapping of Replication Timing Replication timing can be experimentally mapped using nucleotide analogues like bromodeoxyuridine (BrdU). BrdU is incorporated into newly synthesized DNA and can be detected with specific antibodies. By analyzing BrdU incorporation at different time points during S phase, researchers can identify early and late replicating regions of the genome, confirming the temporal program of origin firing and its correlation with chromatin domains. ## Regulation of Replication Initiation: The Pre-Replicative Complex (pre-RC) Eukaryotic cells employ a two-step mechanism to ensure that each replication origin is activated only once per cell cycle. This involves the assembly of an inactive pre-replicative complex (pre-RC) during G1 phase (licensing) and its activation in S phase (origin firing). ### pre-RC Assembly in G1 Phase: Licensing Origins In G1 phase, when Cyclin-Dependent Kinase (CDK1) activity is low, pre-replicative complexes () are assembled at replication origins. This process \"licenses\" origins for replication, ensuring they are competent to initiate replication in the subsequent S phase but prevents premature firing. ### pre-RC Components: ORC, MCM2-7, CDC6, and CDT1 ::: definition **Definition 5** (Pre-Replicative Complex (pre-RC)). *In G1 phase, when Cyclin-Dependent Kinase (CDK1) activity is low, pre-replicative complexes () are assembled at replication origins. This complex licenses origins for replication.* ::: ::: definition **Definition 6** (Origin Recognition Complex (ORC)). *The eukaryotic initiator protein, homologous to DNA A in prokaryotes. ORC binds to throughout the cell cycle, serving as a platform for pre-RC assembly.* ::: ::: definition **Definition 7** (MCM2-7 Complex (MCM2-7)). *The replicative helicase in eukaryotes, homologous to DNA B. The inactive MCM2-7 complex is loaded onto origins during pre-RC formation.* ::: ::: definition **Definition 8** (CDC6 and CDT1). *Helicase loaders, homologous to DNA C. CDC6 and CDT1 are essential for loading MCM2-7 onto ORC at origins during G1 phase.* ::: ### Helicase Inactivation within the pre-RC While CDC6 and CDT1 facilitate MCM2-7 loading, they also maintain MCM2-7 in an inactive state within the pre-RC in G1. These loaders effectively clamp the helicase onto DNA, preventing premature DNA unwinding. ### Activation of Origins at the G1/S Transition The transition from G1 to S phase triggers pre-RC activation and origin firing. This activation is driven by the increased activity of Cyclin-Dependent Kinases (CDK1, specifically CDK11) and Dbf4-dependent kinase (DDK). ### Role of CDK1 and DDK in Origin Firing ::: definition **Definition 9** (Cyclin-Dependent Kinase 1 (CDK1)). *CDK1 activity increases at the G1/S transition and phosphorylates CDC6 and CDT1.* ::: ::: definition **Definition 10** (Dbf4-dependent kinase (DDK)). *DDK is activated in early S phase and phosphorylates MCM2-7 helicases and other pre-RC components.* ::: ### Phosphorylation-Dependent Loader Degradation and Helicase Activation Phosphorylation of CDC6 and CDT1 by CDK1 leads to: 1. **Loader Degradation:** Phosphorylation triggers the degradation of CDC6 and CDT1, preventing further MCM2-7 loading and re-replication at already fired origins. 2. **Helicase Activation:** Phosphorylation events, including MCM2-7 phosphorylation by DDK, activate the MCM2-7 helicase complex. ### Origin Unwinding and Replication Fork Formation Activated MCM2-7 helicases initiate DNA unwinding at the origin, creating a replication bubble. This allows for the recruitment of other replication factors and the establishment of active replication forks. ### Sequential Recruitment of Replication Enzymes Following origin unwinding, replication enzymes are recruited in a specific order. Primase is recruited first to synthesize RNA primers. Subsequently, processive DNA polymerases are recruited to initiate DNA synthesis. *Note: The transcript mentions processive polymerases entering before primase, which is likely an error as primase is required to synthesize the RNA primer for DNA polymerase to initiate extension.* ### Coordination by PCNA and RFC Proliferating Cell Nuclear Antigen (PCNA), the sliding clamp, and Replication Factor C (RFC), the clamp loader, are crucial for eukaryotic replication. PCNA enhances DNA polymerase processivity and, along with RFC, coordinates leading and lagging strand synthesis and chromatin reassembly. # Epigenetic Inheritance during Replication ::: definition **Definition 11** (Epigenetic Inheritance). *Epigenetic inheritance is the process by which epigenetic information, heritable changes in gene expression without alterations to the DNA sequence, is transmitted across cell divisions.* ::: ## Epigenetic Stability and Cellular Identity ### Importance of Epigenetic Stability ::: definition **Definition 12** (Epigenetic Stability). *Epigenetic stability is essential for preserving tissue identity and function.* ::: ### Epigenetic Marks and Tissue-Specific Functions ::: definition **Definition 13** (Epigenetic Marks). *Tissue-specific functions are dictated by distinct patterns of gene expression, which are established and maintained by epigenetic marks, including histone modifications and DNA methylation.* ::: ### Epigenetic Plasticity vs. Stability ::: definition **Definition 14** (Epigenetic Plasticity). *Epigenetic plasticity is the ability to dynamically alter epigenetic marks in response to environmental signals, is also important for development and adaptation.* ::: ## Mechanisms of Histone Modification Inheritance Histone modifications are key epigenetic marks. Their inheritance during DNA replication ensures the propagation of chromatin states and gene expression patterns. ### Semi-Conservative Distribution of Parental Histones ::: definition **Definition 15** (Semi-Conservative Distribution of Parental Histones). *During DNA replication, parental histones are distributed to daughter DNA strands. Specifically, H3-H4 tetramers are distributed semi-conservatively, meaning they are roughly equally segregated to both daughter DNA molecules.* ::: ### Differential Segregation of Histone Subunits H3-H4 tetramers and H2A-H2B dimers exhibit different segregation patterns. H3-H4 tetramers tend to remain associated with DNA and are inherited, while H2A-H2B dimers are moreExchangeable and are replaced with both parental and newly synthesized dimers. This distinction is significant as H3 and H4 carry a substantial portion of pre-existing histone modifications. ### Reader-Writer Complexes for Modification Re-establishment ::: definition **Definition 16** (Reader-Writer Complexes). *Reader-writer complexes function by reading specific histone modifications on parental histones and writing the same modifications on newly deposited histones in proximity.* ::: ### Reader Domains: Bromodomains and Chromodomains ::: definition **Definition 17** (Reader Domains). *Reader domains are protein modules that specifically bind to histone modifications.* ::: ::: definition **Definition 18** (Bromodomains). *Bromodomains recognize and bind to acetylated lysine residues, typically associated with transcriptional activation.* ::: ::: definition **Definition 19** (Chromodomains). *Chromodomains recognize and bind to methylated lysine residues, and in some cases, deacetylated lysine, involved in both transcriptional activation and repression depending on context.* ::: ### Writer Domains: and ::: definition **Definition 20** (Writer Domains). *Writer domains are enzymatic domains that catalyze the addition or removal of histone modifications.* ::: ::: definition **Definition 21** (Histone Acetyltransferases ()). *Histone Acetyltransferases () add acetyl groups to lysine residues, generally associated with transcriptional activation.* ::: ::: definition **Definition 22** (Histone Deacetylases ()). *Histone Deacetylases () remove acetyl groups, typically associated with transcriptional repression.* ::: ### Histone Chaperones in Nucleosome Assembly and PCNA Coordination ::: definition **Definition 23** (Histone Chaperones). *Histone chaperones are essential for nucleosome assembly during replication.* ::: ::: definition **Definition 24** (Nucleosome Assembly Protein 1 (NAP1)). *Nucleosome Assembly Protein 1 (NAP1) facilitates the deposition of H2A-H2B dimers onto DNA.* ::: ::: definition **Definition 25** (Chromatin Assembly Factor 1 (CAF1)). *Chromatin Assembly Factor 1 (CAF1) specifically deposits H3-H4 tetramers onto newly replicated DNA. CAF1 is recruited to replication forks by interacting with PCNA.* ::: ### Histone H1, Nucleophosmin, and Chromatin Compaction ::: definition **Definition 26** (Histone H1). *Histone H1, a linker histone, is crucial for higher-order chromatin compaction (transition from 10nm to 30nm fiber).* ::: ::: definition **Definition 27** (Nucleophosmin). *Nucleophosmin is a chaperone specifically for histone H1, facilitating its incorporation into chromatin.* ::: ### Clinical Relevance: Nucleophosmin in Leukemia Nucleophosmin is an abundant nucleolar and nucleoplasmic protein involved in ribosome biogenesis and histone H1 chaperoning. Mutations in the *NPM1* gene, encoding nucleophosmin, are frequent in acute myeloid leukemia (AML), accounting for approximately 30% of cases. These mutations often lead to aberrant cytoplasmic localization of nucleophosmin and contribute to leukemogenesis, highlighting the functional importance of proper chromatin assembly and epigenetic regulation in preventing disease. # Conclusion This lecture has detailed the mechanisms controlling DNA replication in prokaryotes and eukaryotes, emphasizing the critical role of precise regulation for genome stability and the inheritance of both genetic and epigenetic information. Eukaryotic replication is characterized by multiple origins, temporal control, and a sophisticated licensing system involving the pre-RC to ensure once-per-cycle replication. Furthermore, epigenetic inheritance, particularly the transmission of histone modifications through reader-writer complexes and histone chaperones coordinated with replication, is essential for maintaining cellular identity and tissue homeostasis. **Key Takeaways:** - Prokaryotic replication at `OriC` is regulated by DNA A, ATP hydrolysis, and `GATC` methylation/hemimethylation cycle involving SeqA to prevent re-initiation. - Eukaryotic replication features multiple origins with a temporal firing program linked to chromatin structure and is licensed by pre-RC assembly in G1 and activation by CDK1 and DDK in S phase. - Epigenetic inheritance, especially histone modification inheritance, is crucial for maintaining cell identity and tissue homeostasis. - Reader-writer complexes and histone chaperones like CAF1 and NAP1, coordinated by PCNA, are essential for propagating epigenetic information during replication. **Further Questions and Next Lecture Topics:** The next lecture will address the end-replication problem of linear chromosomes and the role of telomeres and telomerase, along with their implications in aging and cancer. We will also further explore the pathological consequences of dysregulation in replication control and epigenetic inheritance in diseases such as leukemia.