MBB 201 Lecture 7: DNA Replication and Repair

DNA Replication and Repair

Learning Outcomes Overview

• DNA Structure and Organization: Describe the structure and organization of DNA at both the strand and chromosome levels.

• Processes of DNA Replication, Repair, and Recombination: Describe these fundamental genetic processes.

Helpful Breakdown of Learning Outcome (Part 1: DNA Replication)

• Understand the semi-conservative nature of DNA synthesis.

• Interpret and predict the results of the Meselson-Stahl experiment.

• Understand DNA polymerase fidelity in replicating DNA templates.

• Explain the discontinuous synthesis on the lagging strand versus continuous synthesis on the leading strand.

• Describe the steps of DNA synthesis at the replication fork, including involved enzymes and their functions.

• Understand how RNA polymerase primase primes DNA synthesis.

• Understand telomerase function and its connection to cellular aging.

Base-Pairing Enables DNA Replication

• The two strands of the DNA double helix have complementary sequences.

• Each strand can serve as a template to create the other strand.

• DNA replication: The process of making a copy of DNA.

Possible Models of DNA Replication

• Semiconservative: Each daughter molecule contains one parental strand and one newly synthesized strand.

• Dispersive: Daughter molecules have patches of old and new DNA interspersed.

• Conservative: One daughter molecule is entirely new DNA, while the parental double helix remains intact.

Meselson-Stahl Experiment: Determining the Correct Model

• Assay Principle: Bacteria grown in the presence of nitrogen incorporate it into DNA. Using different nitrogen isotopes allows separation of DNA by density.

• Isotopes Used:

  • $\text{HEAVY } (\text{}^{15}\text{N})$

  • $\text{LIGHT } (\text{}^{14}\text{N})$

• Separation Method: $CsCl$ density gradients are used to separate DNA based on its density.

• Experimental Steps and Results:

  1. Initial Growth: E. coli grown for many generations in heavy media ($\text{}^{15}\text{N}$). All bacterial DNA incorporates $\text{}^{15}\text{N}$, resulting in a single heavy band upon centrifugation.

  2. First Generation in Light Media: Cells transferred to light medium ($\text{}^{14}\text{N}$). After one generation, DNA equilibrates at a higher position (lower density). All DNA is a hybrid of one parental ($\text{}^{15}\text{N}$) strand and one daughter ($\text{}^{14}\text{N}$) strand.

     • Both semiconservative and dispersive models would predict a hybrid band after one generation. Conservative would predict separate heavy and light bands.

  3. Second Generation in Light Media: If replication is semiconservative, after a second generation, two bands would be observed:

     • One band corresponding to hybrid DNA ($\text{}^{15}\text{N}-\text{}^{14}\text{N}$).

     • One band corresponding to light DNA ($\text{}^{14}\text{N}-\text{}^{14}\text{N}$).

     • This result definitively supported the semiconservative model because the dispersive model would predict a single band of intermediate density, and the conservative model would predict separate heavy and light bands.

Replication Origins

• DNA replication begins at specific sites called replication origins.

• The double helix is opened at these origins by initiator proteins.

• Replication origins are typically rich in A-T base pairs because A-T pairs have only two hydrogen bonds, making them easier to open than G-C pairs (which have three).

• The number of replication origins varies significantly by species:

  • E. coli (a prokaryote) has 1 origin per genome.

  • Humans (eukaryotes) have 1,000-10,000 origins per genome.

• Having multiple origins allows for faster DNA replication, crucial for organisms with larger genomes.

Replication Forks

• Replication forks are Y-shaped junctions formed at each replication origin.

• Two replication forks are formed at each origin, moving in opposite directions, demonstrating bidirectional DNA replication.

• The location of the replication fork is dynamic, changing as replication proceeds.

Materials Needed for DNA Replication (Requirements)

1. DNA template: The existing DNA strand to be copied.

2. Deoxyribonucleoside triphosphates (dNTPs): The building blocks (A, T, G, C); provide energy.

3. Protein complex: Involving the DNA polymerase enzyme.

4. Primer: An RNA primer is generally required to initiate DNA synthesis.

5. $Mg^{2+}$ ions: Act as a cofactor for DNA polymerase activity.

DNA Replication and Polarity

• DNA is always replicated in a $5' \rightarrow 3'$ direction (nucleotides are added to the $3'$ end).

• Energy for Polymerization: Incoming dNTPs provide the energy.

  • The $3' \text{-OH}$ group of the growing strand attacks the $\alpha$-phosphate of the incoming dNTP.

  • Pyrophosphate is released ($PP<em>i$), which is then rapidly hydrolyzed into two molecules of inorganic phosphate ($2P</em>i$), making the polymerization reaction virtually irreversible.

• Processivity: DNA polymerase remains associated with the DNA and moves along the template strand through many cycles of polymerization without dissociating.

Asymmetrical Replication Forks

• Because the two DNA strands are antiparallel and DNA synthesis always proceeds in the $5' \rightarrow 3'$ direction, the replication fork is asymmetrical.

• Leading Strand: One new strand is synthesized continuously in the direction of the replication fork movement.

• Lagging Strand: The other new strand is synthesized discontinuously in short fragments, known as Okazaki fragments, in the opposite direction of the replication fork movement.

DNA Polymerase: Synthesis and Proofreading

• Enzymatic Function: Catalyzes the addition of nucleotides to the $3'$ end of a growing nucleic acid.

  • Multiple types of DNA polymerases exist in E. coli and eukaryotes.

• Primer Requirement: Requires an existing $3' \text{-OH}$ group to function; it cannot initiate a brand new strand de novo (cannot make DNA from nothing).

• Incredibly Accurate: DNA polymerases exhibit remarkable accuracy due to several contributing factors:

  1. Hydrogen Bonding: Complementary base pairing by hydrogen bonds (e.g., A with T, G with C).

  2. Active Site Monitoring: DNA polymerase monitors base-pairing before catalysis. The active site is shaped to fit only correct base pairs, and correct pairs form noncovalent bonds with the enzyme.

  3. dNTP Concentration: Cell maintains roughly equal concentrations of all four dNTPs.

  4. Proofreading: DNA polymerase has a separate exonuclease site for error correction.

     • If an incorrect nucleotide is added, it is pushed into the exonuclease site.

     • The enzyme removes the incorrect nucleotide via $3' \rightarrow 5'$ exonuclease activity (degrades nucleic acids from the $3'$ end).

RNA Primers for DNA Synthesis

• Since DNA polymerase cannot start a new strand de novo, short lengths of RNA act as primers.

• Primase: An RNA polymerase that synthesizes RNA primers using the DNA template.

  • Unlike DNA polymerase, primase does not have proofreading ability.

  • The RNA primer will eventually be removed.

• Primer Requirements for Strands:

  • Leading Strand: Requires only one primer at the origin.

  • Lagging Strand: Requires one primer for every Okazaki fragment.

Lagging Strand Synthesis

• Lagging strand synthesis occurs in the opposite direction of the replication fork movement.

• To create a continuous new DNA strand from separate Okazaki fragments:

  1. Primer Removal and Replacement: A "repair polymerase" removes the RNA primer using its $5' \rightarrow 3'$ exonuclease activity and replaces it with DNA.

     • This repair polymerase does have proofreading function.

  2. Nick Sealing: The remaining nick (gap) between the newly synthesized DNA and the adjacent Okazaki fragment is sealed by DNA ligase.

DNA Ligase

• Function: Seals gaps (nicks) between two DNA fragments, such as Okazaki fragments during replication.

• Mechanism: Catalyzes the formation of a phosphodiester bond between the $3' \text{-OH}$ end of one fragment and the $5'$-phosphate end of the next.

• Energy Requirement: Requires ATP hydrolysis for its activity.

• Importance: Also crucial for various DNA repair mechanisms.

Proteins at a Replication Fork: The Replication Machine

DNA replication requires the coordinated effort of several proteins to open the double helix and synthesize new DNA. These proteins form a