Week 11

• During replication, the two strands of the parental duplex separate and serve as templates for synthesis of new strands

  
• Replication occurs 5' to 3' just like transcription
• 3' hydroxyl group (OH) attacks the incoming deoxyribonucleotide triphosphate at the innermost phosphate
• Catalyzed by DNA polymerase

  
• Hypothesis: DNA replication is semiconservative: the new molecule will have one parent strand and one newly synthesized strand

  
• Meselson-Stahl experiment in prokaryotes: Labeled DNA with different N (nitrogen) isotopes. The heavier form has an extra neutron (15N) and was used to label the parent strands. After one round of replication in the presence of the lighter form of N, the density of the DNA molecule had intermediate density
• Post-Meselson-Stahl experiment in eukaryotes: Labeled DNA with fluorescent nucleotides. After two rounds of replication, one chromatid strongly fluoresces (dark)

  
• The leading strand has its 3' end pointing toward the replication fork; it is synthesized as one long, continuous molecule as the parental strand is unwound

  
• The lagging strand has its 3' end pointed away from the replication fork; it is synthesized in short, discontinuous (fragmented) pieces
• Enzymes involved in DNA replication


  • Helicase: catalyzes the unwinding of the parental DNA double helix at the replication fork
  • Single-stranded binding proteins: bind to the single-stranded regions of the parental DNA strands and prevent them from coming back together
  • Topoisomerases: relieves the stress of unwinding the DNA double helix at the replication fork
  • DNA polymerase: synthesizes the DNA

  
• Enzymes involved in DNA replication


  • RNA primase: synthesizes a short piece of RNA complementary to the DNA template
  • DNA polymerase: elongates the primer, adding successive DNA nucleotides to the 3' end of the growing strand
  • DNA ligase: catalyzes the joining (ligation) of the discontinuous fragments together

  
• DNA replication begins at many places at once
• Point mutations


  Changes in a single nucleotide

  
• Depending on the consequence i.e. amino acid sequence outcome, these mutations are named differently

  
• Given any scenario where you have a point mutation in a gene and you are told the outcome of the amino acid sequence

  
• Nonsynonymous or missense mutations


  When the amino acid sequence is changed due to the mutation, the protein may fold incorrectly, causing it to no longer function

  
• Mutations can be harmful, be beneficial, or have a neutral effect

  
• Nonsense mutations


  When the codon for the original amino acid is changed to a stop codon translation is terminated which results in a truncated polypeptide

  
• Nearly all truncated proteins are nonfunctional and unstable
• Both types of mutations can cause the protein product to be nonfunctional

  
• Insertions and deletions of several nucleotides

  Frameshift mutations can result in the disruption/shifting of the reading frame

  
• In-frame mutations


  A small deletion or insertion that is an exact multiple of 3 nucleotides results in a polypeptide with fewer (in the case of a deletion) or more (in the case of an insertion) amino acids

  
• In-frame mutations


  • A deletion of three nucleotides eliminates 1 amino acid
  • An insertion of 6 nucleotides adds 2 amino acids
  • The mutant CFTR protein is a result of an in-frame deletion of 3 nucleotides that code for PHE at amino acid 508. The resulting protein does not fold properly

  
• G1 phase


  The first "gap" phase, where the size and protein content of the cell increases in preparation for the S phase. During this phase, many regulatory proteins are made and activated
• S phase


  The "synthesis" phase, where the entire DNA content in the nucleus of the cell is replicated

  
• G2 phase


  The second "gap phase," where the cell prepares for mitosis and cytokinesis
• G0 phase


  Distinguished from the G1 phase because there is no active preparation taking place for cell division. This phase is present in cell types that do not actively divide
• Interphase is G1 phase - G2 phase
• Cyclins


  Proteins that appear and disappear cyclically and regulate cell division

  
• CDKs (cyclin-dependent protein kinases)


  Enzymes that regulate cell division, become active and inactive in cycles
• Cyclin binding induces a conformational change in CDK. CDK alone is inactive. The CDK-cyclin complex is active
• G1/S cyclin-CDK complex


  Active at the end of G1, prepares the cell for S phase
• S cyclin-CDK complex


  Involved in the initiation of DNA synthesis

  
• Enzymes


  • Necessary for cells
  • Role in catalyzing reactions
  • Able to interpret an activation energy graph

  
• M cyclin-CDK complex


  Initiates events associated with mitosis, called the mitosis or maturation promoting factor

  
• Requirements of the Cell


  • A way to encode/transmit information
  • A membrane separating the inside of the cell from the outside
  • ENERGY
• Cyclins undergo ubiquitination, which targets them to be rapidly degraded as the cell cycle progresses

  
• Cell cycle checkpoints


  Cells can pause the cell cycle if something is not right, before progressing to the next stage
• Adenosine Triphosphate (ATP)


  • Stores energy in a form that all cells can readily use to perform the work of the cell
  • Hydrolysis of ATP drives many reactions in cells

  
• When chemical reactions occur, bonds between atoms are broken and new bonds are formed