DNA Replication, Repair, and Recombination Notes

DNA Replication, Repair, and Recombination

Copying the Genome

  • Before a cell divides, it must copy its DNA to share with the next cell.
  • Copying the human genome takes approximately 8 hours (S-phase).
  • This is equivalent to copying "Essential Cell Biology" 1000 times.
  • The process makes only one or two mistakes, highlighting its high fidelity.

DNA as a Template

  • DNA strand serves as a template for its own duplication.
  • Preferential binding occurs between base pairs (A&T, G&C), enabling each strand to act as a template for forming its complementary strand.
  • S can serve as a template to generate S’.
  • S’ can serve as a template to generate S.

DNA Replication

  • DNA Replication: Production of two complete double helices from the original DNA molecule.
  • Each new strand is identical to the parent strand.
  • Semi-Conservative: One old strand and one new strand.
  • Original strands remain intact for many generations.
  • DNA replication is "semi-conservative."

DNA Synthesis

  • DNA helix must be opened to expose unpaired bases (unwound).
  • Initiator proteins break hydrogen bonds, separating a short length of DNA.
  • Replication origins (Ori’s): Sites at which DNA is first opened.
    • Bacterial genome: single replication origin.
    • Eukaryote: several replication origins (shortens time).
Initiator Proteins
  • Recognize specific sequences of DNA (replication origin).
  • Break hydrogen bonds to pry two strands apart.
  • A-T rich regions are typically found at replication origins.

Replication Forks

  • Two Y-shaped junctions (replication forks) per origin.
  • Replication forks move away in opposite directions from multiple replication origins in a eukaryotic chromosome.
  • Orange = parental DNA strand; Red = newly synthesized DNA.

Replisome

  • Replisome: The replication machine.
  • Contains all the proteins needed to copy the DNA.
  • Copies DNA at a rate of 100 nucleotide pairs per second in humans.

DNA Helicase

  • Separates and opens DNA strands so that proteins/enzymes have access to the genetic materials.
  • Utilizes ATP hydrolysis to pry apart the double helix.
  • Spins like a motor to unravel the chromatin structure, "opening" the DNA.

DNA Polymerase

  • Synthesizes new DNA using an old strand of DNA as a template.
  • Only synthesizes in the 5’-to-3’ direction!
  • Stays associated with DNA and moves along the template strand for many cycles of polymerization.
DNA Synthesis Direction
  • DNA is synthesized in the 5’ to 3’ direction.
  • Releases pyrophosphate (PPi) following linkage.
  • Nucleotides enter the reaction as nucleoside triphosphates, which provide the energy for polymerization.
  • =\phosphoanhydride bond!
  • At the replication fork, the two newly synthesized DNA strands are of opposite polarities.

Okazaki Fragments

  • DNA is synthesized in the 5' to 3' direction

Primase, Nuclease, Repair Polymerase, and DNA Ligase

  • Primase: An RNA polymerase that generates a short length of RNA (about 10 nucleotides in length) - the primer!
    • Provides a base-paired 3’ end as a starting point for DNA polymerase.
  • Nuclease: Breaks apart RNA primer.
  • Repair Polymerase: Replaces RNA with DNA.
  • DNA Ligase: Joins 5’ phosphate of new DNA to adjacent 3’ hydroxyl end of the next DNA.
  • DNA is synthesized in the 5’ to 3’ Direction.

Leading and Lagging Strand DNA Synthesis

  • Leading-strand replication is continuous.
  • Lagging-strand replication is discontinuous; RNA primers are removed, and Okazaki fragments are joined together by DNA ligase.

DNA Polymerase Error Rate & Proofreading

  • DNA polymerase makes an error in about 1 in every 10710^7 base pairs.
  • When a rare mistake is made and a wrong nucleotide is added, it is corrected using proofreading.
  • Proofreading occurs in the 3’ to 5’ direction (copy editing!).
  • DNA Polymerase has separate sites for synthesis and proofreading.
  • Coordination of these two domains is why protein synthesis only occurs 5’ to 3’!

PCNA

  • Proliferating cell nuclear antigen (PCNA) is a “donut”-shaped molecule that enhances DNA synthesis elongation by associating with DNA polymerase.
  • Sliding clamp (PCNA) promotes elongation.

RP-A

  • RP-A (Replication Protein A) binds to single-strand DNA, protects it from nucleases, and “straightens out” any secondary structures.
  • Single-strand binding protein (SSB) or RP-A (human) binds to ss DNA.

Replication Machine & Clamp Loader

  • Replication Machine Uses ATP Hydrolysis
  • Prevents DNA from re-forming base pairs & keeping DNA elongated to serve as template
  • Keeps DNA attached to template
  • Clamp loader (PCNA) - hydrolyzes ATP to lock clamp around DNA
  • Proteins are held together in a large replication machine at the Ori
  • The replication machine at the Ori

Topoisomerases

  • During DNA replication, torsional tension is created ahead of the replication fork.
  • Becomes overwound, and DNA replication could halt!
  • Parental and daughter DNA molecules are also tangled together.
  • Topisomerase I and II resolve tension and tangled DNAs by making single- or double-stranded breaks in phosphate backbone.
  • Act on the topology of DNA
Topoisomerase I and II
  • Topoisomerase I makes a ”nick” in one strand and relieves torsional stress.
  • Topoisomerase II makes a double-strand DNA break and untangles DNA molecules.
  • Some antibiotics and chemo drugs target topo I and II to block DNA replication
    • Topo I = Camptothecin
    • Topo II = Doxorubicin
    • Topo II in bacteria = Ciprofloxacin

Telomerase

  • Reach end of DNA - no location to lay down RNA primer needed for Okazaki fragment
  • Specific telomere sequences attract telomerase – Humans telomere sequence – TTAGGG – Use RNA template that is part of the enzyme
Telomeres and Telomerase action
  • Telomerase binds to the template strand.
  • Telomerase adds additional telomere repeats to the template strand (RNA-templated DNA synthesis).
  • Completion of lagging strand by DNA polymerase (DNA-templated DNA synthesis).
  • Forms End of Chromosome.

Importance of Learning DNA Replication

  • Basis of many lab techniques!
    • PCR
    • Sequencing (Sanger, Illumina, Etc.)

DNA Damage and Mutation

  • Mutation - permanent change in the DNA
  • DNA Repair
  • Accumulation of mutations leads to cancer (aging!)

Mis-match Repair

  • Mis-match repair is directly linked to DNA synthesis
  • During Replication mistake occurs.
  • Replication without repair leads to mutated DNA molecule.
MutS/MutL Complex
  • distorts geometry of double helix
  • Makes a single-stranded break
  • Mis-match repair is directly linked to DNA synthesis
  • MutS/MutL complex on DNA
  • Nick = newly synthesized strand (different cells have different strategies for recognizing new strand)

Base Damage

  • Base damage is the most common type of damage.
    • Oxidative damage
    • Hydrolytic attack
    • Uncontrolled methylation
  • Spontaneous DNA damage could be as high as 10,000 events per cell and day
  • Leads to a base change!
  • Leads to a base loss (happens to adenine as well)

Thymine Dimers

  • Sunlight (UV) causes thymine dimers.
  • Covalently link adjacent thymine bases

Excision Repair

  • Step 1: Nuclease cleaves covalent bonds that join damaged base or nucleotides to rest of strand
    • Nuclease specific to type of DNA damage
  • Step 2: Repair DNA polymerase binds 3’ hydroxyl end and fills in gap
    • 5’-to-3’ direction
    • Same proofreading activity
  • Step 3: DNA ligase seals the nick
  • Base, nucleotide and single strand break repair work this way

DNA Damage Response

  • Some proteins serve as “communicators” or “sensors” important for determining cell fate after DNA damage (ATM kinase)

Double-Strand Break Repair

  • Double strand breaks very toxic to cell (and genome)
Non-homologous End-Joining (NHEJ)
  • Usually alters the original DNA by deletions or insertions.
  • FAST and EASY
Homologous Recombination Repair (HRR)
  • More complicated and less frequent but is precise
  • SLOW and DIFFICULT
  • Uses the “other” chromosome as a template for “perfect” repair
  • Double-strand break is accurately repaired

Human Diseases Associated with Defective DNA Repair

  • Examples:
    • Ataxia Telangiectasia: ATM (damage sensor); HRR NHEJ; Cancer predisposition (very young), Extreme IR sensitivity
    • Xeroderma pigmentosum: XPA; NER; sensitive to UV light (sunlight)
    • Cockayne Syndrome: ERCC6 ERCC8; NER; Sensitivity to UV, early aging
    • Fanconi Anemia: FANC family (13+ genes); HRR; Cancer predisposition, short stature
    • Nijmegen Breakage Syndrome: NBS (damage sensor); HRR; Immune deficiency, IR sensitive, cancer
    • Werner Syndrome: WRN (a helicase); HRR; Accelerated aging
    • Bloom Syndrome: BLM; HRR; Immune deficiency, cancer, early aging
    • Hereditary non-polyposis Colorectal cancer: MLH1, MSH2, MSH6, PMS1, PMS2; MMR; Increased incidence of colorectal cancer and other cancers
    • Li-Fraumnei: p53; NER HRR; Increased cancer incidence
    • Trichothiodystrophy: NER; Short stature, brittle hair, intellectual impairment
  • Werner syndrome
  • Ataxia Telangiectasia
  • BRCA 1/2 defects (tumor suppressor proteins)

Cellular Response to DNA Damage

  • DNA damage from endogenous or exogenous agents
  • Triggers sensors and signalling pathways
  • Modulation of metabolism
  • Salvage pathways
  • Chromatin remodeling
  • Gene expression
  • Protein PTMs (post-translational modifications)
  • Protein synthesis
  • Protein degradation
  • Protein translocation
  • Nuclear export/import
  • DNA repair
  • Cell cycle arrest
  • Cell death pathways
  • Apoptosis
  • Stress responses

ATM Kinase

  • ATM kinase senses DNA damage and amplifies the signal to the cell