DNA replication and repair

Abstract

Cell division involves the duplication of the 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. The duplication of DNA (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 at each single strand. Two double DNA strands are formed, each with 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, such as the incorporation of the wrong base, still occur. External factors as well as internal cellular processes lead to alterations in the chemical structure of DNA. If DNA errors are not repaired, mutations and/or cell destruction may occur. DNA repair mechanisms are thus important to ensure a sufficient degree of genomic stability.

DNA replication

Fundamentals of DNA replication

  • Purpose: To ensure that daughter cells contain genetically identical information to the parent cells by copying double-stranded DNA (dsDNA) during cell division (of the S phase of the cell cycle)
  • DNA replication:
    • Is semiconservative: Replication results in two identical dsDNA molecules, each consisting of a parent strand (serves as a template) and a newly synthesized daughter strand.
    • Occurs in the 5' → 3' direction
    • Is bidirectional: Replication occurs in both directions of the original dsDNA (i.e., the direction of each parent strand).
    • Occurs in three stages :
      • Initiation
      • Elongation
      • Termination
    • Involves many proteins/enzymes

Proteins involved in DNA replication

Protein involved Task Prokaryotes Eukaryotes
Helicase Unwind local segments of the DNA double helix (ATP-dependent reaction) at the replication fork DnaB MCM complex
Single-stranded DNA-binding proteins (SSBs) Prevents reannealing of separated strands SSB (single-strand binding protein) RPA (replication protein A)

Topoisomerase

Type I topoisomerase

Cleaves only one of both DNA strands

  • Has a nuclease (cuts the strand) and a ligase (reseals the strand)
  • Does not require ATP
Topoisomerase I (type IA topoisomerases) Topoisomerase I (type IB topoisomerases)
Type II topoisomerase

Temporarily cleaves both DNA strands for larger structural alterations of the DNA

Topoisomerase II (DNA gyrase) and topoisomerase IV

Topoisomerase II

Primase (DNA-dependent RNA polymerase) Synthesis of short RNA sequences (RNA primers) Primase (DnaG) Primase activity as a component of DNA polymerase α
DNA-dependent DNA polymerase Extends RNA primers by adding 50–100 nucleotides Polymerase α
Replicates the lagging strand DNA polymerase III Polymerase δ
Extend the leading strand Polymerase ε
Removal of primers RNase H and DNA polymerase I (5'→3' exonuclease activity) RNase H and FEN-1 (flap endonuclease-1)
Gaps between fragments are filled after primer removal DNA polymerase I Polymerase δ
DNA clamp A subunit of DNA polymerase that anchors polymerase to the DNA β-subunit of DNA polymerase III PCNA (proliferating cell nuclear antigen)
Ligase Links newly synthesized DNA fragments (ATP or NAD+-dependent reaction) by catalyzing the formation of phosphodiester bonds DNA ligase DNA ligase
Replisome The complex of proteins (i.e., DNA polymerase, helicase, etc.) that builds to participate in DNA replication Replisome Replisome
Telomerase Ensures complete replication of the ends of linear chromosomes Telomerase

The process of DNA replication

1. Initiation

  1. Specific proteins (comprising a prepriming complex) recognize and bind to the origin of replication
  2. At the ori, helicase separates and begins unwinding dsDNA into single strands, forming 2 replication forks
  3. SSBs prevent the single strands from reannealing and protects ssDNA from cleavage
  4. Supercoil relaxation: DNA topoisomerases relieve overwinding (positive supercoils) or underwinding (negative supercoils) that develop during DNA separation and elongation.

2. Elongation

  1. Primer synthesis: : Primase synthesizes a short, 5–10 nucleotide long RNA primer that is complementary to the template strand
  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 as 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 and remove incorrectly paired nucleotides.
  4. Primer removal: RNA primers are excised in the opposite direction of synthesis (so 5'→3')
    1. In prokaryotes, by RNase H and 5'→3' exonuclease of the DNA polymerase I
    2. In eukaryotes, by 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.
    1. In prokaryotes, DNA polymerase I adds deoxynucleotides one at a time and “proofreads”
    2. In eukaryotes, by Polymerase δ
  6. Joining of the ends

DNA synthesis results in the nucleophilic attack of the 3'OH group of the growing DNA strand to the α-phosphate of the incoming deoxyribonucleotide triphosphate!

3. Termination

  • Termination of replication is initiated by binding termination proteins to termination sequences.
  • There are various termination mechanisms for circular and linear DNA molecules.

Telomeres

  • Structure: a non-coding DNA fragment of several thousand bp (composed of tandem repeats of TTAGGG) at the 3' ends of chromosomes
  • Function
  • Maintenance: by telomerase
    • Telomerase
      • A special reverse transcriptase that carries its own RNA template (ribonucleoprotein) and can elongate telomeres
      • Has a strong presence in frequently dividing cells (to prevent loss of function). Not present in all cells!
      • Activity is enhanced in cancer cells

Mechanisms of DNA damage

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

Endogenous sources of DNA errors

Exogenous sources of DNA errors

  • Toxic substances
    • Alkylating substances: methylate or ethylate bases, causing them to pair with other bases than the usual
      • For instance, 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: embed between the stacked DNA base pairs, causing replication to stop and increasing the risk of strand breaks
  • Radiation
    • UV radiation (both UVA and UVB) can result in dimer formation of neighboring pyrimidine bases (pyrimidine dimers)
      • Thymine dimers are mainly formed, linked by a cyclobutane ring.
      • Dimers create bulky helix distortions that interfere with DNA replication, which increase the risk of developing mutations (e.g., BRAF gene mutation in melanoma).
    • Ionizing radiation
      • Can lead to ssDNA and dsDNA breaks
    • Also results in the increased formation of free radicals

Typical causes of DNA damage:
Deamination of cytosine to uraciluracil pairs with adenine!
UV radiation → thymine dimers!
Ionizing radiationfree radical formation → DNA damage!

DNA repair mechanisms

Type of DNA repair Mechanisms Notable conditions if defective
ssDNA repair Base excision repair
  1. Base-specific glycosylase (DNA glycosylase) recognizes and removes damaged base (deaminated or oxidated), creating an AP site
  2. AP endonuclease cuts the DNA backbone on the 5' end of the AP site
  3. AP lyase cuts the DNA backbone on the 3' end of the AP site
  4. DNA polymerase-beta refills the resulting gap
  5. Ligase reseals the strand
  • Cancers
Nucleotide excision repair
  1. Specific endonucleases recognized the damaged area (e.g., dimer formation) of nucleotides (typically a 12–24-bp section)
  2. Oligonucleotide containing the damaged region is excised by exonuclease
  3. DNA polymerase refills the resulting gap
  4. Ligase reseals the strand
DNA mismatch repair
  1. The newly synthesized (unmethylated) strand is differentiated from the methylated parent strand
  2. Mismatched base pairs are recognized by MSH proteins
  3. The damaged daughter strand is cut by endonuclease
  4. Damaged nucleotides are removed by exonuclease
  5. DNA polymerase refills the resulting gap
  6. Ligase reseals the strand

dsDNA break repair

Nonhomologous end joining
  1. 2 DNA fragments ends are combined via DNA ligase IV
  2. 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
  3. Possible throughout the whole cell cycle
Homologous end joining
  1. Repair by exchanging homologous segments between two DNA molecules (sister chromatids)
  2. Late S phase or G2 phase
  3. The error can be repaired using complementary strand in the sister chromatid, meaning it is
  4. Requires an identical (or nearly identical) sequence to serve as a template for repair

Base excision repair is a BAADL (Base-specific glycosylase, AP endonuclease, AP lyase, DNA polymerase-beta, Ligase)

last updated 10/22/2018
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