DNA replication and repair

Summary

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.

DNA replication

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

Protein Function Enzyme in prokaryotes Enzyme in eukaryotes
Helicase
  • MCM (minichromosome maintenance) complex
Single-stranded DNA-binding protein (SSBs)
  • SSB

Topoisomerase

Topoisomerase I
  • Cleaves only one of both DNA strands
  • Has nuclease activity to cut DNA strands
  • And has ligase activity to reseal the ligated strand
  • Does not require ATP
Topoisomerase II
  • Cleaves both DNA strands for larger structural alterations of DNA
  • Requires ATP
Primase (DNA-dependent RNA polymerase)
DNA-dependent DNA polymerase
  • N/A
  • Polymerase α
  • DNA polymerase III: inhibited by certain drugs (e.g., antiretrovirals) via chain termination (the modified 3′-OH group of the drug prevents further elongation of the nucleotide chain)
  • Polymerase δ
  • Extends the leading strand (until it reaches the preceding fragment's primer)
  • 5′→3′ synthetic activity
  • 3′→5′ proofreading exonuclease activity
  • Polymerase ε
  • Removal of primers (by replacing them with a DNA fragment)
  • Gaps between fragments are filled after primer removal
  • Polymerase δ

Ligase

  • Links newly synthesized DNA fragments (Okazaki fragments) by catalyzing the formation of phosphodiester bonds
  • ATP or NAD+-dependent reaction

Telomerase

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
  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
  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.
  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: RNA primers are excised in the opposite direction of synthesis (5'→3' direction)
  5. Filling the gaps: During primer removal, DNA polymerase fills the gaps with deoxynucleotides complementary to the parent strand until the free ends meet.

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.”

Telomeres

  • 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
    • 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!

Mechanisms of DNA damage

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

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

DNA repair mechanisms

Type of DNA repair Phase of cell cycle Mechanisms Associations with defective repair
ssDNA repair
Base excision repair
Nucleotide excision repair
DNA mismatch repair
dsDNA break repair
Nonhomologous end joining
  • DNA that has undergone a double-stranded break is repaired via DNA ligase IV.
  • Short homologous sequences called microhomologies comprise the single-stranded tails of the DNA ends to be joined.
    • Repair is usually accurate if microhomologies are compatible
    • Nonhomologous end joining is itself prone to errors and mutagenic defects because no error-free template is available.
    • Some DNA may be lost in the process.
Homologous recombination
  • Repair by exchanging homologous segments between two DNA molecules (sister chromatids)
  • The error can be repaired using the complementary strand from the intact sister chromatid.
  • Requires a (nearly) identical sequence (such as the complementary strand) to serve as a template for repair
  • No DNA is lost in the process.

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.

  • 1. Bregje van Oorschot, Giovanna Granata, Simone Di Franco, Rosemarie Ten Cate, Hans M Rodermond, Matilde Todaro, Jan Paul Medema, Nicolaas A P Franken. Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment. Oncotarget. 2016. url: https://pubmed.ncbi.nlm.nih.gov/27602767/.
  • 2. Susan S. Wallace, Ph.D, Drew L. Murphy, Ph.D., and Joann B. Sweasy, Ph.D. Base Excision Repair and Cancer. Cancer Letters. 2012. url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361536/.
last updated 11/18/2020
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