BIOCH 330 Midterm

• DNA replication in bacteria:

  • DNA polymerase enzyme copying the parental DNA strand guided by the RNA primer
  • Helicase enzyme unwinding the DNA double helix
  • Single-stranded binding protein (SSB) keeping the strands separated
  • Clamp loader complex helping to hold the DNA polymerase in place
  • Termination factor (Ter) responsible for terminating the replication process
• Types of DNA replication:

  • Conservative: strands remain intact, parent structure stays intact, progeny synthesized
  • Semiconservative: strands remain intact, parent structure hybrid with new strand
  • Dispersive: strands don't remain intact, have both new and old portions evenly
• Meselson and Stahl's experiment:

  • Used centrifugation to differentiate between replication mechanisms
  • Grew bacteria in N15 enriched media to create higher density DNA
  • Cesium chloride gradient centrifugation allowed separation of samples by density
  • Showed semi-conservative replication occurred, with intermediates where N14 was blended
• Replication proceeds from the 5' to 3' end of the new strand
• Template DNA runs 3' to 5'; the 3' OH is deprotonated by Mg and acts as an electrophile that attacks the nucleotide triphosphate, making PPi the leaving group that can be converted to 2P with pyrophosphatase
• 2 ATP are used for every base pair added
• Replication fork: where replication occurs, is bidirectional and moves apart
• Replication bubble consists of two oppositely moving replication forks, with one strand replicated simultaneously as the leading strand and the other in fragments as the lagging strand with Okazaki fragments
• Primers allow the extension of the synthesized strand and are synthesized with the action of separate enzymes like primase, which doesn't require an existing 3' OH group
• Polymerase enzymes involved in replication combine 5' to 3' synthesis with exonuclease activities and are metalloenzymes that have Mg2+ promote nucleophilic attack from 3' OH to deprotonate the O-
• DNA Polymerase III is the primary enzyme of replication, while DNA Polymerase I is critical in lagging strand synthesis and repair
• Exonuclease 3'-5' activities allow for proofreading and removes non watson crick base pairs and try to add proper nucleotides
• Polymerase structure: Klenow fragments of Pol I was the first discovered polymerase and does not include 5' to 3' exonuclease activity the DNA binding site is positively-charged it has "fingers and thumb" orientation with a palm that is connected to 3'-5' exonuclease
• DNA replication differs in eukaryotic cells as it is coordinated with the cell cycle and has multiple replication origins
• Eukaryotic DNA Polymerase:

  • DNA polymerase α is associated with primase
  • DNA polymerase δ needs association with PCNA for highly processive lagging strand synthesis
  • DNA polymerase ε is highly processive for leading strand synthesis
• DNA polymerase α has no 3' to 5' exonuclease activity and is associated with primase. Has moderate processivity with no PCNA, it is included in initiation and associated with primers and has no proof reading
• DNA polymerase δ has 3'-5' exonuclease activity. Doesn't associate with primase and has high processivity where PCNA is required. Is Analogous to B clamp and ensures DNA association while proofreading lagging strand
• Mutations are permanent changes in the sequence of bases caused by radiation damage or spontaneous chemical reactions
• Mutations can be mutagenic if replicated and are grouped into three types: transitions, transversions, and insertions/deletions
• Radiation damage can lead to mutations, with adjacent thymine residues forming dimers that prevent replication
• Deamination is the hydrolytic removal of an amino group from a base. Cytosine is converted to Uracil by removing the amino group and adding Oxygen. It is easy to repair but can form uncommon base pairs and replication of it results in a transition mutations
• Replication of deamination can result in transition mutations, and oxidation of guanine can generate 8-oxoguanine that forms non-strandard interaction with A that is a transversion mutation
• Alkylation- some methylations affect base pairs while others don't. May be involved in protein-DNA interactions
• Depurination is the spontaneous hydrolysis of the glycosidic bond, affecting purines and creating an apurinic site
• Insertions and deletions can be caused by intercalating agents or larger scale recombination processes
• Direct repair mechanisms involve returning modified bases to their original state
• Thyminedimers are repaired by reversion of covalent bonds
• Alkylation is repaired by transferring alkyl groups off nucleotides to other molecules
• Photolyases repair damaged DNA by reverting back to the base form in the presence of visible light
• Base Excision Repair involves DNA glycosylase removing a bad base, creating an AP site, AP endonuclease cutting the phosphodiester backbone to the AP site, polymerase filling gaps and removing extra residues, and DNA ligase sealing the phosphodiester bond
• E. coli uses UvrA, UvrB, and UvrC proteins to bind to pyrimidine dimers, cut the backbone, and remove a 13-base section
• Humans have a similar repair mechanism using 16 proteins to remove 30 nucleotides
• Xeroderma Pigmentosa is a condition of extreme UV sensitivity due to defective nucleotide excision repair in humans
• N1 in pyrimidines binds to C1' in sugar while N9 in Purines binds to C1' in Sugar
• Adenine is a purine and contains one substituent off C6 and it is a NH2 group
• Guanine is a purine that has two subsitutients an Oxygen off C6 and a NH2 group off C2
• Cytosine is a pyrimidine with two substituents an oxygen off C2' and NH2 off C4
• Uracil is a pyrimidine with two substituents, 2 Oxygens on C2 and C4
• Thymine is a pyrimidine with 3 substituents two oxygens off C2 and C4 and a Methyl group off C5
• Primary structures are held together by Covalent bonds and 3'-5' phosphodiester linkages the phosphates add an overall negative charge.