DNA Replication and Repair: Mechanisms and Regulation

DNA Replication and Repair

DNA Replication

DNA replication is a fundamental process in all living cells, ensuring the accurate duplication of genetic material before cell division. The process involves several key steps and enzymes to guarantee fidelity and efficiency.

A. DNA Replication is Semi-Conservative

• Semi-conservative replication means that each new DNA molecule consists of one original (parental) strand and one newly synthesized strand.

• This mechanism was demonstrated by the Meselson-Stahl experiment.

B. DNA Synthesis is Bidirectional

• Replication begins at specific origins and proceeds in both directions, forming two replication forks.

• In prokaryotes (e.g., E. coli), the circular chromosome has a single origin, resulting in two identical circular chromosomes after replication.

• In eukaryotes, multiple origins of replication exist on each linear chromosome, allowing rapid and efficient DNA synthesis.

C. Unwinding DNA Strands

• DNA helicase: Unwinds the double helix at the replication fork.

• Topoisomerase: Relieves supercoiling ahead of the replication fork.

• Single-stranded binding proteins (SSB): Stabilize unwound DNA strands and prevent re-annealing.

D. DNA Synthesis Proceeds in the 5' → 3' Direction

• DNA polymerase adds nucleoside triphosphates (dATP, dGTP, dCTP, dTTP) to the 3' end of the growing DNA strand.

• Two phosphates are removed from each nucleotide to provide energy for phosphodiester bond formation.

• All known DNA polymerases synthesize DNA in the 5' to 3' direction.

E. The RNA Primer

• DNA polymerases cannot initiate synthesis de novo; they require a short RNA primer synthesized by primase.

• Primase adds a short RNA sequence complementary to the DNA template.

• DNA polymerase extends the primer by adding DNA nucleotides to its 3' end.

• Later, RNA primers are removed and replaced with DNA by DNA polymerase I (in prokaryotes), and the gaps are sealed by DNA ligase.

F. DNA Replication has a Leading and Lagging Strand

• Leading strand: Synthesized continuously in the direction of the replication fork movement.

• Lagging strand: Synthesized discontinuously as short fragments called Okazaki fragments, which are later joined by DNA ligase.

• Replication is always 5' to 3', so the lagging strand must be synthesized in segments as the fork opens.

G. Proofreading

• DNA polymerase III (in prokaryotes) and DNA polymerases δ and ε (in eukaryotes) possess 3' to 5' exonuclease activity for proofreading.

• This activity removes incorrectly paired nucleotides, ensuring high fidelity of DNA replication.

H. Telomerase and Telomere Replication

• Telomerase is a ribonucleoprotein enzyme that extends the 3' end of linear chromosomes (telomeres) in eukaryotes.

• It carries an RNA template complementary to the telomeric repeat sequence, allowing extension of the parental DNA strand.

• Prevents loss of genetic information at chromosome ends during replication.

• Telomerase activity is high in germ cells and stem cells, but low in most somatic cells.

DNA Repair

DNA repair mechanisms are essential for maintaining genome stability by correcting errors and damage that occur during replication or from environmental sources.

A. Mutations

• Mutation: A permanent change in the DNA sequence.

• Spontaneous mutations can occur during DNA replication.

• Tautomer formation: Rare forms of bases can mispair, leading to mutations.

• Damage can also occur due to hydrolysis (e.g., deamination) or chemical agents.

• Deamination: Loss of an amino group from a base (e.g., cytosine to uracil).

B. Mutagens

• Chemical mutagens: Base analogues, base-modifying agents, and intercalating agents can alter DNA structure and pairing.

• Radiation: UV light induces thymine dimers; ionizing radiation causes strand breaks and base modifications.

C. DNA Repair Mechanisms

• Excision repair: Removes damaged bases or nucleotides and replaces them with correct ones.

• Types of excision repair:

  • Base excision repair (BER): Repairs small, non-helix-distorting base lesions.

  • Nucleotide excision repair (NER): Removes bulky, helix-distorting lesions (e.g., thymine dimers).

• Mismatch repair: Corrects errors missed by DNA polymerase proofreading, such as mispaired bases and small insertion/deletion loops.

• In E. coli, the mismatch repair system distinguishes the parental strand by DNA methylation.

• DNA ligase seals the final phosphodiester bond after repair synthesis.

Table: Comparison of DNA Repair Mechanisms

Repair Mechanism	Main Function	Key Enzymes	Type of Damage Repaired
Base Excision Repair (BER)	Removes small, non-helix-distorting base lesions	DNA glycosylase, AP endonuclease, DNA polymerase, DNA ligase	Deaminated bases, oxidized bases
Nucleotide Excision Repair (NER)	Removes bulky, helix-distorting lesions	Excinuclease, DNA polymerase, DNA ligase	Thymine dimers, bulky adducts
Mismatch Repair (MMR)	Corrects replication errors not fixed by proofreading	MutS, MutL, MutH (in bacteria), DNA polymerase, DNA ligase	Mismatched bases, small insertions/deletions

Additional info:

• DNA methylation can affect gene expression and is used by repair systems to distinguish old and new DNA strands.

• Defects in DNA repair pathways can lead to genetic diseases and increased cancer risk (e.g., xeroderma pigmentosum from NER defects).