bio 101 - m6

• Semiconservative

  • Proved by Meselson and Stahl (1958) using heavy (H) and light (L) isotopes of N
• Origin of Replication

  • Rich in AT pairs
• Replisomes - Collection of proteins and enzymes involved in the complex process of replication
• Initiation Proteins of E. coli: DnaA
• Initiation proteins of yeast: ORC protein
• DNA Helicases - requires ATP to denature DNA helix, binds ssDNA
• Helicase II - attaches to template for lagging strand, moves 5' → 3'
• Rep protein - attaches to template for leading strand, moves 3' → 5
• DNA helicase moves along one DNA strand only
• Single-strand DNA Binding Proteins - Helix destabilizing proteins, bind to sites where template has been unwound but not yet duplicated
• In eukaryotes, major SSBP is Replication Protein A
• Primases - Generates the RNA primers, Short chain polyribonucleotide (4-12 nt) which rovides the 3’ –OH group needed in DNA chain elongation
• enzymatic activities of DNA polymerase: 5'->3' polymerization activity
• Processivity - property of DNA pol to remain attached to the template and incorporate nucleotides before detaching
• Fidelity - 1 mistake per 109 or 1010 ntds added
• 3'->5' exonuclease activity - proofreading
• 5'->3' exonuclease activity - removes RNA primers at the 5' end, not present in eukaryotic DNA polymerase
• In E. coli, there are five DNA polymerases
• polA - Major repair enzyme
• polB - Minor repair enzyme
• polC - Replicase
• Bacterial DNA Polymerase III Holoenzyme - Replicates both strands simultaneously, An aggregate of 10 proteins
• alpha - core enzyme for 5’→3’ polymerization
• epsilon - core enzyme for 3’→5’ exonuclease /proofreading
• theta - core enzyme stimulates epsilon
• beta2 - sliding clamp
• sliding clamp - keeps DNA polymerase on to allow duplication of long stretches of DNA
• clamp loader - Uses ATP to assemble the sliding clamp to the DNA
• Processivity is achieved by the sliding clamp
• 5’→3’ exonuclease activity of DNA POL I can be removed by treating DNA pol I with the protease subtilisin
• Small fragment: 5'->3' exonuclease activity
• Klenow fragment - Large fragment With retained polymerase and 3’→5’ exonuclease activity
• Klenow Fragment - Can be used in in vitro synthesis of ds DNA from ssDNA
• Eukaryotic Proliferating Cell Nuclear Antigen - Analogous to the E. coli sliding clamp
• DNA Ligase - Seals nicks using NAD and ATP
• Topoisomerases - Relaxes DNA; removes DNA supercoils
• topoisomerase II - involved in releasing the final products of circular DNA replication