DNA replication

Main Themes:
​ Maintaining DNA Fidelity: Cells go to great lengths to ensure accurate DNA replication and
repair. This high fidelity is crucial for individual survival and long-term species evolution.
​ Complexity of DNA Replication: DNA replication is a sophisticated process involving numerous
protein machines working in a coordinated manner.
​ Eukaryotic vs. Prokaryotic Replication: While sharing fundamental principles, DNA replication
differs between eukaryotes and prokaryotes in terms of complexity, regulation, and the
challenges presented by linear chromosomes.
Key Ideas and Facts:
1. Maintaining DNA Sequences:
​ Low Mutation Rates: Mutations, permanent changes in DNA sequence, can be detrimental.
Organisms have evolved mechanisms to maintain remarkably low mutation rates (approximately
1 change per 10^10 nucleotides per cell generation).
​ "Although E. coli and humans differ greatly in their modes of reproduction and in their
generation times, when the mutation rates of each are normalized to a single round of DNA
replication, they are both extremely low and within a factor of three of each other."
​ "The cells of a sexually reproducing animal or plant are of two types: germ cells and somatic
cells. The germ cells transmit genetic information from parent to offspring; the somatic cells
form the body of the organism. We have seen that germ cells must be protected against high
rates of mutation to maintain the species."
​ Importance of Low Mutation Rates: High mutation frequencies would lead to:
​ Limits on the number of essential proteins an organism can utilize, hindering complexity.
​ Increased incidence of cancer due to rapid accumulation of mutations in somatic cells.

Timeline of DNA Replication

This timeline details the process of DNA replication as described in the provided source, focusing
primarily on the events at the replication fork:
1. Initiation:
​ Origin Recognition: Initiator proteins bind to specific DNA sequences at the replication origin,
rich in A-T base pairs.
​ Helix Destabilization: The initiator protein complex wraps the DNA, weakening the double helix.
​ Helicase Loading: Two DNA helicases, bound to helicase loaders, are attracted to the origin.
​ Primer Synthesis: The helicases unwind the DNA, allowing primase to synthesize short RNA
primers.
​ Replication Fork Assembly: Remaining proteins, including DNA polymerases, assemble to form
two replication forks moving in opposite directions.
2. Elongation:
​ Leading Strand Synthesis: DNA polymerase continuously synthesizes the leading strand in the
5'-to-3' direction.
​ Lagging Strand Synthesis: DNA polymerase synthesizes the lagging strand discontinuously in
the 5'-to-3' direction, creating Okazaki fragments.
​ Okazaki Fragment Priming: DNA primase synthesizes RNA primers for each Okazaki fragment.
 ​ Sliding Clamp Action: Sliding clamps keep DNA polymerase attached to the DNA during
elongation.
​ Proofreading: DNA polymerase proofreads its work using a 3'-to-5' exonuclease activity,
removing mismatched nucleotides.
​ Primer Removal and Gap Filling: RNA primers are removed by a repair enzyme, and the gaps are
filled by DNA polymerase.
​ Fragment Joining: DNA ligase seals the nicks between Okazaki fragments, creating a
continuous lagging strand.
​ Nucleosome Assembly: New nucleosomes are assembled behind the replication fork, using a
combination of parental and newly synthesized histones.
3. Termination:
​ Fork Collision: Replication forks collide, completing the replication of the entire chromosome.
​ Replication Machine Disassembly: The replication machinery is disassembled.
4. Telomere Replication:
​ Telomerase Action: Telomerase extends the 3' end of the lagging strand template using its own
RNA template.
​ Lagging Strand Completion: DNA polymerase completes the lagging strand using the
telomerase extension as a template.
​ Telomere Protection: Specialized proteins bind to the telomere, forming a protective cap
(shelterin) and potentially forming t-loops to further shield the chromosome ends.
​
Cast of Characters
This section lists the principle actors involved in DNA replication:
1. DNA Polymerase:
​ Function: Catalyzes the addition of nucleotides to the growing DNA strand during replication.
​ Key Features:Synthesizes DNA in the 5'-to-3' direction.
​ Possesses proofreading exonuclease activity for error correction.
​ Requires a primer to initiate synthesis.
2. DNA Primase:
​ Function: Synthesizes short RNA primers that provide the 3'-OH group required for DNA
polymerase to start synthesis.
​ Key Features:Can initiate RNA synthesis de novo without a primer.
​ Essential for lagging strand synthesis.
3. DNA Helicase:
​ Function: Unwinds the DNA double helix ahead of the replication fork, separating the two
strands.
​ Key Features:Uses ATP hydrolysis for energy.
​ Moves along one of the DNA strands.
​ Essential for exposing the template strands for replication.
4. Single-Strand DNA-Binding Proteins (SSB):
​ Function: Bind to single-stranded DNA, preventing the formation of hairpins and keeping the
template strands accessible for replication.
​ Key Features:Stabilize unwound DNA.
​ Prevent reannealing of the separated strands.
​ Facilitate DNA polymerase binding.
5. DNA Ligase:
 ​ Function: Seals the nicks in the DNA backbone, joining Okazaki fragments on the lagging strand
and completing the sugar-phosphate backbone.
​ Key Features:Requires ATP for energy.
​ Creates a phosphodiester bond between adjacent nucleotides.
6. DNA Topoisomerase:
​ Function: Relieves torsional stress and DNA tangling ahead of the replication fork.
​ Key Features:Type I: Creates transient single-strand breaks to allow DNA rotation.
​ Type II: Creates transient double-strand breaks to allow DNA strand passage.

Role of DNA Topoisomerases:

● Type I Topoisomerase: Creates a temporary single-strand break, allowing the DNA to rotate and
relieve strain. It does not require additional energy as the original bond energy is stored
temporarily, allowing for easy reformation of the phosphodiester bond.
● Type II Topoisomerase: Works by breaking both DNA strands, forming a gate that allows another
helix to pass through. ATP hydrolysis is used to reset the enzyme and close the gate. This
enzyme can prevent entanglement, particularly useful during cell division.
​
7. Initiator Proteins:
​ Function: Bind to replication origins and initiate the assembly of the replication machinery.
​ Key Features:Recognize specific DNA sequences at the origin.
​ Promote the recruitment of helicases and other replication proteins.
8. Sliding Clamp:
​ Function: Holds DNA polymerase onto the DNA template, increasing processivity and speed.
​ Key Features:Ring-shaped structure encircles the DNA.
​ Loaded onto DNA by a clamp loader protein.
​ On the leading strand, the clamp remains associated with the polymerase for a long time.
​ On the lagging strand, the clamp and polymerase dissociate after each Okazaki fragment, and a
new clamp is loaded for the next fragment.
​
9. Clamp Loader:
​ Function: Loads the sliding clamp onto DNA at the primer-template junction.
​ Key Features:Uses ATP hydrolysis for energy.
​ Recognizes the primer-template junction.
10. Mismatch Repair System:
​ Function: Corrects errors that escape proofreading by DNA polymerase.
​ Key Features:Recognizes and removes mismatched nucleotides.
​ Distinguishes the newly synthesized strand from the parental strand to ensure accurate repair.
11. Telomerase:
​ Function: Extends the ends of linear chromosomes, maintaining telomere length.
​ Key Features:Contains an RNA component that serves as a template for DNA synthesis.
​ A type of reverse transcriptase.
12. Histones and Histone Chaperones:
​ Function: Histones package DNA into nucleosomes, and histone chaperones facilitate the
assembly of new nucleosomes behind the replication fork.
​ Key Features:Histones: Basic proteins that form octamers around which DNA wraps.
​ Histone Chaperones: Bind histones and regulate their assembly onto DNA.
13. Shelterin:
​ Function: Protein complex that binds to telomeres, protecting them from being recognized as
DNA damage and preventing chromosome fusions.
​ Key Features:Binds to the specific repetitive DNA sequence of telomeres.
​ May help form t-loops, further shielding chromosome ends.
​
Initiation and Completion of DNA Replication in Chromosomes:
​ Replication Origins: Specific DNA sequences where helix opening and replication initiation
occurs.
​ Bacteria:Single origin of replication.
​ Tightly regulated initiation to ensure one round of replication per cell division.
​ Eukaryotes:Multiple origins of replication per chromosome.
​ Replication occurs during S phase of the cell cycle.
​ Different regions on the same chromosome replicate at distinct times.
​ "The different replication origins in these eukaryotic chromosomes are activated in a sequence,
determined in part by the structure of the chromatin, with the most condensed regions of
chromatin typically beginning their replication last."
​ Nucleosome Assembly: New nucleosomes are assembled behind the replication fork using both
parental and newly synthesized histones.
​ Telomeres:Specialized structures at the ends of linear chromosomes, preventing DNA loss
during replication.
​ Replicated by telomerase, a unique enzyme with an RNA template.
​ "Eukaryotes solve it in a different way: they have specialized nucleotide sequences at the ends
of their chromosomes that are incorporated into structures called telomeres."
​ May play a role in regulating cell lifespan and aging.
​
​ Eukaryotic Replication Speed: Eukaryotic replication forks move more slowly than bacterial
forks due to the presence of nucleosomes, which must be bypassed or remodeled for
replication.

1. DNA Replication in E. coli

● Key Proteins:
○ Initiator: dnaA protein.
○ Helicase: dnaB protein.
○ Primase: dnaG protein.
● Initiation:
○ Multiple dnaA proteins bind to specific DNA sequences at the origin.
○ Binding destabilizes the double helix by wrapping DNA around the protein.
○ Helicases are recruited with the help of helicase-loading proteins (dnaC proteins) that
inhibit helicase activity until proper loading.
○ Single-strand binding proteins stabilize the opened DNA, allowing primases to
synthesize primers.
● Replication Forks:
○ Two replication forks form and proceed in opposite directions.
○ The rate of bacterial replication forks is significantly faster (~20x) than in eukaryotes due
to simpler packaging requirements.
2. Eukaryotic DNA Replication

​ General Characteristics:
○ DNA replication occurs only in the S phase of the cell cycle.
○ Eukaryotic replication forks move at approximately 50 nucleotides per second, slower
due to tightly packed chromatin.
○ Multiple origins of replication (30,000–50,000 in humans) are activated in a
coordinated, sequential manner.
​ Mechanism:
○ Each origin is activated only once per cell cycle to ensure accurate genome replication.
○ DNA origins are recognized by the ORC (origin recognition complex) and other
associated factors.
○ Replication begins with helicase loading (Mcm helicase in eukaryotes).
​ Cell Cycle Regulation:
○ During G1 phase, a prereplicative complex (including ORC and helicases) forms at each
origin.
○ Entry into S phase activates these complexes via kinase phosphorylation.
○ Phosphorylation of ORC prevents new prereplicative complex formation, ensuring each
origin fires only once per cycle.

​
Mutation Rates in Bacteria and Humans:

● Bacteria: ~1 mutation per 10610^6106 generations.

● Humans: ~1 mutation per 10810^8108 nucleotides per generation.

Why is DNA replication considered a high-fidelity process?

DNA replication boasts incredibly high fidelity, meaning it produces very few errors. This is achieved
through several proofreading mechanisms:
​ Precise Base Pairing: Correct nucleotides have a higher affinity for the DNA polymerase,
making them more likely to be incorporated.
​ Conformational Change: DNA polymerase undergoes a shape change that double-checks
the base pair geometry before adding a nucleotide.
​ Exonucleolytic Proofreading: If an incorrect nucleotide is added, DNA polymerase has a
separate site that removes the mismatched nucleotide, allowing the process to continue
correctly.

DNA Proofreading and Fidelity

​ High Accuracy: DNA polymerase has proofreading capabilities, significantly reducing

errors during replication. Only one mistake occurs per 101010^{10}1010 nucleotides.
​ Proofreading Steps:
 ○ Initial Selection: DNA polymerase prefers correct base pairs due to energetically
favorable pairing.
○ Conformational Change: The enzyme tightens around the active site if the base
pair is correct, further reducing errors.
○ Exonucleolytic Proofreading: If an incorrect nucleotide is added, the 3' to 5'
exonuclease activity removes the mismatch, allowing correct base-pairing.
○ 5′ to 3′ Polymerization: Initial synthesis with error rate 1 in 10510^5105.
○ 3′ to 5′ Exonuclease Activity: Corrects mismatches with an additional error rate
reduction of 1 in 10210^2102.
○ Strand-Directed Mismatch Repair: Further error correction, reducing the error rate
by 1 in 10310^3103.

​ Strand-Directed Mismatch Repair: A separate system scans newly replicated DNA,

identifying and correcting errors missed by the DNA polymerase.

Strand-Directed Mismatch Repair

1. Mechanism in Bacteria:
○ Errors missed by proofreading exonuclease are corrected by mismatch repair.
○ New DNA strands are identified by unmethylated adenine in GATC sequences,
leading to selective error correction on new strands.
2. Mechanism in Eukaryotes:
○ Newly synthesized strands contain nicks that signal repair proteins to correct
replication errors selectively.
​

How are new nucleosomes assembled during DNA

replication?
1. Nucleosome Duplication During DNA Replication

● DNA Replication and Nucleosome Disassembly: As the DNA is being replicated, the
double helix unwinds and the parental strands are separated. Nucleosomes, which are made
of histones and DNA, are disrupted during this process.
● Histone H3-H4 Tetramers: The histone proteins H3 and H4 form tetramers (two H3-H4
dimers joined together). During replication, these tetramers are retained on the parental DNA
strands. However, they do not remain tightly bound. They are loosely associated with the
DNA and are not fully disassembled.
● Histone H2A-H2B Dimers: The histone dimers H2A-H2B, on the other hand, detach from
the DNA completely during replication. These dimers need to be replaced as the nucleosome
structure is reformed after replication.
2. Histone Chaperones and Their Role

● Histone Chaperones: These are proteins that help in the assembly and incorporation of
histones onto the newly synthesized DNA strands. Histones do not bind directly to the DNA
by themselves; instead, chaperones facilitate their proper placement.
● NAP1 and CAF1: Two key histone chaperones are NAP1 and CAF1.
○ NAP1: This chaperone primarily helps load histones H2A and H2B onto the newly
replicated DNA.
○ CAF1: This chaperone plays a crucial role in the assembly of the H3-H4 tetramers on
the newly synthesized DNA strands.
● PCNA (Proliferating Cell Nuclear Antigen): PCNA is a sliding clamp that aids in the
replication process. It helps guide the histone chaperones NAP1 and CAF1 to the DNA by
interacting with them. PCNA ensures that histones are added efficiently to the new DNA in a
timely manner.

3. Nucleosome Reassembly

● Once the histones are added to the DNA by the chaperones, the newly incorporated histones
(H2A-H2B dimers and H3-H4 tetramers) assemble into functional nucleosomes.
● The newly synthesized DNA becomes wrapped around these histone complexes, forming
nucleosomes, just like the parental DNA. This is crucial for maintaining the structure and
function of the genome during cell division.

What are telomeres and why are they important?

Telomeres are repetitive DNA sequences found at the ends of eukaryotic chromosomes. They
protect the chromosome ends from degradation and fusion and solve the end-replication problem of
linear chromosomes. The enzyme telomerase maintains telomere length by adding new repeats to
the 3' end of the DNA strand.
How is telomere length regulated and what are the
consequences of telomere shortening?
Telomere length is regulated by a balance between the actions of telomerase and natural
degradation processes. In many somatic cells, telomeres gradually shorten with each cell division,
eventually leading to replicative cell senescence. This may act as a safeguard against uncontrolled
cell proliferation and cancer. However, excessive telomere shortening can also lead to genetic
instability and contribute to aging and age-related diseases.

DNA Repair and Recombination Mechanisms

1. Repair Mechanisms: Cells have various repair systems to correct DNA damage,
enhancing genetic stability.
2. Homologous Recombination: A repair method that uses a sister chromatid as a template,
ensuring accurate repair of double-strand breaks.
3. Transposable Elements: Special DNA sequences that move within the genome and play a
role in genetic diversity and evolution.