Detailed Notes on DNA Replication

DNA Duplex Orientation

• DNA strands are antiparallel, oriented in opposite directions.
• One strand runs 5' to 3', the complementary strand runs 3' to 5'.

Base Pairing Rules

• Adenine (A) pairs with Thymine (T).

• Guanine (G) pairs with Cytosine (C).

• When strands separate, each serves as a template for a new strand.

• Enzymes bring in complementary nucleotides to match the template strand.

  • For example, if a template strand has the sequence T-G-C-A, the new strand will be A-C-G-T.

DNA Replication Hypotheses

• Three proposed models for how DNA replication occurs:

  • Semiconservative
  • Conservative
  • Dispersive

Semiconservative Replication

• Proposed by Watson and Crick.
• DNA strands separate, each serving as a template.
• Each new DNA duplex contains one parental strand and one newly synthesized strand.

Conservative Replication

• Parental duplex remains intact after replication.
• Intermediate molecule formed with parental and new strands.
• Parental strands rejoin, and new strands form a new duplex.

Dispersive Replication

• Parental duplex is fragmented.
• New duplexes contain a mix of parental and new DNA segments.
• No intact parental strands are conserved.

Meselson and Stahl Experiment

• Performed in the 1950s at Caltech by Meselson and Stahl.
• Demonstrated that DNA replication is semiconservative.

Experimental Procedure

• Bacteria were grown in a medium containing the heavy isotope Nitrogen-15 ($N^{15}$
• DNA of these bacteria became heavy due to the incorporation of $N^{15}$. This is called "heavy DNA".
• Bacteria were then transferred to a medium containing the lighter isotope Nitrogen-14 ($N^{14}$).
• Bacteria were allowed to replicate for one or two generations in the $N^{14}$ medium.
• DNA was extracted and analyzed after each generation.

Predictions Based on Replication Models

• Using gray to represent $N^{15}$ strands and red for $N^{14}$ strands.

Conservative Replication Prediction

• After one round of replication in $N^{14}$ medium:

  • Two distinct populations of DNA: one heavy ($N^{15}$$N^{15}$) and one light ($N^{14}$$N^{14}$). The parental strand rejoins and the new strand is lighter.

• After two rounds of replication in $N^{14}$ medium:

  • Parental DNA reappears and two brand new molecules. Still two distinct populations of DNA.

Semiconservative Replication Prediction

• After one round of replication in $N^{14}$ medium:

  • Single hybrid DNA: each duplex contains one heavy ($N^{15}$) and one light ($N^{14}$) strand. The parental strand splits up and winds up one half of it in one new duplex.

• After two rounds of replication in $N^{14}$ medium:

  • Two types of DNA: hybrid DNA (one $N^{15}$ and one $N^{14}$ strand) and light DNA (both $N^{14}$ strands).

Centrifugation in Cesium Chloride

• Used to separate DNA based on density.
• Heavier DNA settles near the bottom of the centrifuge tube.
• Lighter DNA settles near the top.
• Hybrid DNA settles in between.

Experimental Results

• Parental DNA ($N^{15}$$N^{15}$) formed a band at the bottom of the tube.
• After one replication in $N^{14}$, a single band appeared, lighter than the parental band.
• After two replications in $N^{14}$, two bands appeared: one at the hybrid position and one at the lighter position ($N^{14}$$N^{14}$).
• These results supported the semiconservative model.

Disproving Conservative Replication

• Conservative replication would have resulted in two bands after one replication: one heavy and one light.
• This was not observed.

Disproving Dispersive Replication

• After one round, dispersive and semiconservative appear the same, hybrids are formed.
• After two rounds, dispersive would have one band. This band would continue to get lighter with additional rounds.
• The scientists were able to confirm semiconservative after the second round because of the distinct band of $N^{14}$.

Basic Concept of Semiconservative Replication

• Parental strands separate and serve as templates.
• Nucleotides are brought in and paired up with the template strands.
• Sugar-phosphate backbone is sealed to form new strands.
• Hydrogen bonds hold the strands together.
• Each new duplex contains one parental and one new strand.
• Since the two strands of DNA are complementary, each strand provides the information to act like a template for building a new strand in replication.

Timing of DNA Replication

• Occurs during the Synthesis (S) phase of the cell cycle.
• S phase can last around eight hours in a 24-hour human cell cycle.
• G1 phase about 11 hours.
• G2 phase about 4 hours.
• Mitosis about 1 hour.

Rate of Replication

• Typical human chromosome contains about 50,000,000 nucleotide pairs.

• DNA polymerase (enzyme that makes DNA polymer) moves slowly.

  • Eukaryotes DNA polymerase: ~50 nucleotides per second
  • Prokaryotes DNA polymerase: ~500 nucleotides per second

• At this rate, it would take 800 hours to replicate a typical chromosome, which is why replication starts at multiple sites simultaneously (origins of replication).

Origins of Replication

• Multiple origins of replication exist in the human genome (thousands).
• Not every cell uses the same origins.
• Proteins bind to these sites and initiate the melting of hydrogen bonds.
• Eukaryotic chromosomes start replication at multiple origins simultaneously, but they don't all start at the same time.