Cell division involves the duplication of a cell's entire DNA so that two genetically identical daughter cells arise from a single cell. DNA is bound to proteins in the nucleus and is tightly packed. Therefore, DNA replication requires that the DNA is loosened and the double helix is unwound. Specific proteins, including DNA polymerase, then synthesize a complementary daughter strand of double-stranded DNA (dsDNA) from each single strand template. This leads to the formation of two dsDNA molecules, each composed of one new and one original strand. The process of DNA replication includes control mechanisms to keep the genetic information as stable as possible, but errors (e.g., the incorporation of a wrong base) still occur. External factors as well as internal cellular processes lead to alterations in the chemical structure of DNA. Unrepaired DNA errors can cause mutations and/or cell destruction. DNA repair mechanisms are, therefore, important to ensure genomic stability.
Fundamentals of DNA replication
- Purpose: the process of copying dsDNA during the S phase of cell division, ensuring the transmission of identical genetic information from the parent cell to the daughter cell.
- Is semiconservative: Replication results in two identical dsDNA molecules, with each new molecule of dsDNA consisting of a parent strand (which serves as the template strand) and a newly synthesized daughter strand.
- Takes place in the 5′ → 3′ direction
- Is bidirectional:
- Begins at multiple points
- Occurs in three stages :
- Initiation (See “The process of DNA replication” in this section below.)
|Protein||Function||Enzyme in prokaryotes||Enzyme in eukaryotes|
|Single-stranded DNA-binding protein (SSBs)|| || || |
|Primase (DNA-dependent RNA polymerase)|| || |
|DNA-dependent DNA polymerase|| || |
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The process of DNA replication
Origin of replication (ori): a specific DNA sequence in the genome where DNA replication starts
- Specific proteins (comprising a prepriming complex) recognize and bind to the origin of replication.
- The origin of replication contains AT-rich sequences (e.g., TATA box regions), similar to those found in promoters
- Prokaryotes with circular genomes have a single origin.
- Eukaryotes have numerous oris (e.g., 30,000–50,000 in humans) at which DNA replication begins simultaneously.
- Replication fork: Y-shaped region in the chromosome where both leading and lagging strands are replicated from the DNA template
- Prevention of reannealing: SSBs prevent the single strands from reannealing and protect ssDNA from cleavage.
- Supercoil relaxation: DNA topoisomerases relieve overwinding (positive supercoils) or underwinding (negative supercoils) that develop during DNA separation and elongation.
- Primer synthesis
DNA synthesis: For simultaneous replication of both parent strands, DNA replication occurs continuously on the leading strand and discontinuously on the lagging strand in a 5′→3′ direction. At the same time, complementary deoxynucleotides are added to the free 3′-OH group of the daughter strand.
Reaction catalyzed by DNA polymerase (polymerase δ in eukaryotes, DNA polymerase III in prokaryotes)
- 5′→3′ DNA polymerase activity catalyzes the nucleophilic attack of the 3′-OH group on the α-phosphate of the incoming deoxynucleotide triphosphate, forming an ester bond.
- DNA strands are elongated by one nucleotide at a time.
- Pyrophosphate (PPi) is released.
- Pyrophosphate is cleaved into two phosphates by pyrophosphatase.
Leading strand: a complementary daughter strand of DNA whose replication is continuous due to its free 3′-OH end
- The free 3′-OH end is initially on the primer.
- Only one primer is required.
- Lagging strand: a complementary daughter strand of DNA whose replication is discontinuous because new RNA primers are constantly being synthesized at the moving replication fork to ensure a free 3′-OH end for elongation
- Reaction catalyzed by DNA polymerase (polymerase δ in eukaryotes, DNA polymerase III in prokaryotes)
- Proofreading: Some polymerases (e.g., DNA polymerase I and III) have ; 3′→5′ exonuclease activity to proofread recently synthesized DNA and to remove incorrectly paired nucleotides.
Primer removal: RNA primers are excised in the opposite direction of synthesis (5'→3' direction)
- DNA polymerase I and δ have 5′→3′ exonuclease activity that enables the enzymes to remove the RNA primer.
- In prokaryotes; , by RNase H and 5′→3′ exonuclease activity of DNA polymerase I. In eukaryotes, the RNA primer is displaced by DNA polymerase δ and excised by the enzyme FEN-1 (flap endonuclease-1)
Filling the gaps: During primer removal, DNA polymerase fills the gaps with deoxynucleotides complementary to the parent strand until the free ends meet.
- In prokaryotes, DNA polymerase I replaces the RNA primer by adding deoxynucleotides one at a time and proofreads as it does so.
- In eukaryotes, polymerase δ replaces the RNA primer.
- DNA ligase joins (ligates) the free ends of Okazaki fragments, resulting in a continuous strand of DNA.
- Ligase reaction
- Initiated by binding termination proteins to termination sequences
- There are various termination mechanisms for circular and linear DNA molecules (e.g., a termination site sequence in the DNA).
- Structure: : a noncoding DNA fragment of several thousand base pairs (composed of tandem repeats of TTAGGG) at the 3′ end of chromosomes
Prevention of structural gene loss during replication of linear DNA double-strands
- The lagging strand becomes shorter with each process of DNA replication.
- The leading strand can be completely synthesized.
- Telomeres prevent the continuous loss of nucleotides from the 3′ end of DNA during replication and raise the number of possible replication cycles that a given cell can undergo.
- Prevention of structural gene loss during replication of linear DNA double-strands
- Maintenance by telomerase: a type of reverse transcriptase that carries its own RNA template (ribonucleoprotein) and can elongate telomeres
To remember the TTAGGG sequence added by the telomerase: Tell Them All: Genes Gotta Go!
Endogenous sources of DNA damage
- Repetitive sequences: DNA polymerase is error-prone in reading repetitive DNA sequences. The repetitive region is repeatedly read, especially in triplet repeats.
- Keto-enol tautomerism: a transient tautomeric shift of nucleotide bases from the usual keto-configuration to the enol-configuration, which can result in mismatched base pairing (e.g., enol-thymine pairs with guanine instead of adenine)
- Depurination: thermal cleavage of the N-glycosidic bond between deoxyribose and a purine base at body temperature
- Free radical damage: highly reactive oxygen or hydroxyl radicals that cause oxidative stress and can chemically alter bases
Spontaneous of DNA bases causes incorrect base pairing (mismatching)
- Deamination of cytosine to uracil (e.g., as a result of nitrite uptake in food), which then pairs with adenine
- Deamination of 5-methylcytosine to thymine, which pairs with adenine instead of guanine.
- Deamination of adenine to hypoxanthine
- Deamination of guanine to xanthine
- Alkylating substances
- Intercalating substances
- UV radiation (both UVA and UVB) can result in dimer formation of neighboring pyrimidine bases (pyrimidine dimers)
- Mainly causes dsDNA breaks, but can also cause ssDNA breaks 
- Also results in increased formation of free radicals
|Type of DNA repair||Phase of cell cycle||Mechanisms||Associations with defective repair|
|Base excision repair|| || |
|Nucleotide excision repair|
|DNA mismatch repair|| |
|dsDNA break repair|
|Nonhomologous end joining|| || |
|Homologous recombination repair|| |