DNA replication and repair

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.
• DNA replication:
  • 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:
    • Replication occurs on both strands of the original dsDNA simultaneously in opposite directions; (i.e., the 5′→ 3′ direction of each parent strand).
    • Two active replication forks are formed at the site of DNA double-strand separation.
  • Begins at multiple points
  • Occurs in three stages :
    • Initiation (See “The process of DNA replication” in this section below.)
    • Elongation
    • Termination

Proteins involved in DNA replication

Helicase divides DNA into two Halves. Ligase Ligates the Okazaki fragments!

The process of DNA replication

Initiation

1. 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.
2. Replication fork: Y-shaped region in the chromosome where both leading and lagging strands are replicated from the DNA template
   • Helicase separates and begins unwinding dsDNA into single strands at the ori, forming two replication forks.
   • Replication occurs on both strands simultaneously (bidirectionally), but always in a 5′ → 3′ direction.
3. Prevention of reannealing: SSBs prevent the single strands from reannealing and protect ssDNA from cleavage.
4. Supercoil relaxation: : DNA topoisomerases relieve overwinding (positive supercoils) or underwinding (negative supercoils) that develop during DNA separation and elongation.

Elongation

1. Primer synthesis
   • RNA primers: a 10–12 nucleotides long RNA sequence that is complementary to the template strand
     • Synthesized by RNA primase
     • Provide a 3′-OH group to which DNA polymerases can attach a DNA nucleotide.
   • The RNA primer is removed at a later stage.
2. 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 (PP_i) 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
     • Okazaki fragments; : discontinuous segments of 1,000–2,000 nucleotides that arise during lagging strand creation
       • Each DNA segment requires its own RNA primer.
       • Each primer is synthesized at the moving replication fork.
3. 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.
4. Primer removal:
   • 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)
5. 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
       • DNA ligase transfers an AMP residue to the 5′ phosphate end of one of the DNA fragments to be bound.
       • AMP is cleaved and the 5′ phosphate end is bound to the 3′-OH end of the other fragment.

Termination

• 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).

DNA replication inhibitors have a modified 3′-OH end that prevents the elongation of the existing nucleotide chain, a phenomenon also known as “chain termination.”

• Structure: : a noncoding DNA fragment of several thousand base pairs (composed of tandem repeats of TTAGGG) at the 3′ end of chromosomes
• Function
  • Prevention of structural gene loss during replication of linear DNA double-strands
    • The lagging strand becomes shorter with each process of DNA replication.
      • DNA polymerase cannot synthesize the last part of the 5′ end of the lagging strand because there is no 3′-OH group present to fill the gaps following removal of the RNA primer.
      • A section of the telomere is lost instead of the coding segment of the DNA.
    • 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.
• Maintenance by telomerase: a type of reverse transcriptase that carries its own RNA template (ribonucleoprotein) and can elongate telomeres
  • Only present in eukaryotic cells (especially in fast-dividing cells such as stem cells)
  • Activity is enhanced in cancer cells
  • Adds a DNA polymer (TTAGGG) to avoid loss of genetic material from the 3′ end of the DNA strand with every replication

To remember the TTAGGG sequence added by the telomerase: Tell Them All: Genes Gotta Go!

There are endogenous or exogenous sources of DNA damage, which may result in mutations and/or cell death if not repaired.

Endogenous sources of DNA damage

• Replication errors
  • 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)
• Chemical instability
  • Depurination: thermal cleavage of the N-glycosidic bond between deoxyribose and a purine base at body temperature
    • An apurinic/apyrimidinic site (AP site) is formed.
    • AP sites belong to the most common physiologically occurring DNA lesions (estimate of > 10,000/day and per cell in eukaryotes).
  • Free radical damage: highly reactive oxygen or hydroxyl radicals that cause oxidative stress and can chemically alter bases
    • Example: guanosine to 8-oxoguanosine
    • Result: During replication, 8-oxoguanosine pairs with adenine and not cytosine.
  • Spontaneous deamination 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
      • Uracil is usually recognized as an error and replaced with cytosine through base excision repair.
      • If this does not occur, uracil pairs with adenine instead of cytosine with guanine during the next replication.
    • Deamination of 5-methylcytosine; to thymine, which pairs with adenine instead of guanine.
    • Deamination of adenine to hypoxanthine
    • Deamination of guanine to xanthine

Exogenous sources of DNA errors

• Toxic substances
  • Alkylating substances
    • Mechanism of action: methylate or ethylate bases cause incorrect base pairing, e.g., O⁶-ethylguanine is formed by the alkylation of guanine and pairs with thymine instead of cytosine.
    • Examples: mustard gas, N-nitrosodimethylamine, dimethyl sulfate
  • Intercalating substances
    • Mechanism of action: embedding between the stacked DNA base pairs, causing replication to stop and increasing the risk of strand breaks
    • Examples: ethidium bromide, acridine dye, dactinomycin
• Radiation
  • UV radiation (both UVA and UVB) can result in dimer formation of neighboring pyrimidine bases (pyrimidine dimers)
    • Mainly formation of thymine dimers, linked by a cyclobutane ring
    • Dimers create bulky helix distortions that interfere with DNA replication, which stops replication prematurely, increasing the risk for further mutations (e.g., BRAF gene mutation in melanoma).
  • Ionizing radiation
    • Mainly causes dsDNA breaks, but can also cause ssDNA breaks ^[1]
    • Also results in increased formation of free radicals

Enzymes of base excision repair: GEL PLease = Glycosylase, AP-Endonuclease, Lyase, DNA Polymerase, DNA Ligase

Excision repair occurs in the G1 phase of the cell cycle. DNA mismatch repair occurs in the S phase of the cell cycle.