DNA Repair

Secondary Structure of RNA

  • RNA is more flexible compared to DNA due to the presence of 2’OH.

  • Capable of forming various structures:

    • Bends

    • Loops

    • Bulges

    • Hairpins

    • Helical segments

  • Watson-Crick pairing rules apply:

    • Adenine (A) pairs with Uracil (U) instead of Thymine (T).

Classes of RNA

Transfer RNA (tRNA)

  • Smallest RNA molecules.

  • Comprised of a single strand that folds back into a common shape, despite sequence variations.

Ribosomal RNA (rRNA)

  • Larger than tRNAs (e.g., 23S, 16S, and 5S in prokaryotes).

  • Folds into structures essential for ribosome function:

    • Common shape with regions of divergence and conservation across kingdoms.

Nucleases

  • Catalyze the hydrolysis of phosphodiester bonds in nucleic acids.

  • Types include:

    • Deoxyribonucleases (DNases): Target DNA.

    • Ribonucleases (RNases): Target RNA.

    • Exonucleases: Degrade nucleic acids from their ends.

    • Endonucleases: Cleave nucleic acids internally.

Endonucleases and Their Function

  • Cut internal phosphodiester bonds.

  • Restriction Endonucleases: Recognize palindromic sequences.

    • Examples of recognition and cleavage sites:

      • EcoRI (5' GAATTC 3')

      • BamHI (5' GGATCC 3')

      • HindIII (5' AAGCTT 3')

  • Enzyme names reflect their bacterial origin.

Origin of Restriction Endonucleases

  • Produced by bacteria to defend against phage infections.

  • DNA methylation protects bacterial DNA while phage DNA is left unmodified, making it vulnerable to cleavage.

Organization of DNA in Cells

  • DNA is organized into chromatin through interactions with proteins.

  • Nucleosome: Smallest unit of chromatin, consisting of histone proteins.

    • Constructed of 2 copies of H2A, H2B, H3, and H4.

    • Forms a "beads on a string" structure leading to chromatid and chromosome formation.

DNA Replication and Repair

Basics of DNA Replication

  • Essential for genetic information transmission during cell division.

  • Major understanding drawn from studies in prokaryotes, particularly E. coli.

Models of DNA Replication

  • Three hypotheses:

    1. Conservative: Parental strands re-associate after replication.

    2. Semi-conservative: Each new strand contains one original and one new strand (supported by experimental evidence).

    3. Dispersive: Parent strands are dispersed into two new molecules.

Density Gradient Centrifugation

  • Method to separate DNA using a cesium chloride gradient to analyze density post-replication.

Complementary DNA Sequences

  • Complementary sequence to a given strand is determined by base pairing (A-T, G-C).

Synthesis and Function of DNA Polymerases

  • DNA polymerase I: Catalyzes nucleotide addition in 5’ to 3’ direction, using free 3’OH.

  • Exonuclease activity: Corrects mismatched bases during replication.

Lagging and Leading Strand Synthesis

  • Lagging strand synthesis occurs in fragments (Okazaki fragments) due to opposite orientation to leading strand.

  • DNA replication initiates with RNA primers synthesized by primase.

Sealing Nicks in DNA

  • Nicks in the DNA backbone are sealed by DNA ligase once all fragments are joined.

DNA Repair Mechanisms

Types of Mutations

  • Spontaneous Mutations: Resulting from normal cellular processes.

  • Induced Mutations: Resulting from external agents or mutagens.

Categories of Single-Nucleotide Mutations

  • Substitutions: One nucleotide replaces another.

    • Transitions: Purine to purine or pyrimidine to pyrimidine.

    • Transversions: Purine to pyrimidine or vice-versa.

  • Insertions: Addition of extra nucleotides.

  • Deletions: Removal of nucleotides.

Mismatch Repair in E. coli

  • Nicked strands are degraded by exonucleases and template is filled by DNA polymerases.

Base Excision Repair

  • Mechanism for repairing DNA that removes bases damaged or misincorporated properties.

Double Strand Break Repair

  • Tumor suppressor gene BRCA1 functions in double-strand break repair, mutations linked to breast cancer susceptibility.

DNA Gel Electrophoresis

Procedure

  • Agarose gel used for DNA separation, electrical current applied to move negatively charged DNA toward the anode.

Size Estimation

  • Mobility plotted against size standards helps estimate fragment size.

The Central Dogma of Molecular Biology

  • Flow of genetic information:

    • DNA ➔ RNA (Transcription) ➔ Protein (Translation).