Lecture 17 Flashcards
DNA Replication
What is the Griffith Experiment (1920s)?
Earliest evidence that DNA may be the hereditary information of the cell. Used mice and streptococcus pneumoniae.
What is the Avery, Macleod, and McCarty Experiment (1940)?
Test to see if DNA, RNA or protein is the causative agent of the “transformation” effect observed by Griffith.
Who discovered DNA? When was DNA discovered?
Watson, Crick, and Franklin discovered the structure of DNA. DNA’s double-helix structure was discovered in 1950s.
What is the structure of DNA?
5 carbon sugar (2-deoxyribose), with a phosphate group attached to the 5’ carbon of the sugar, and a nitrogenous base attached to 1’ carbon of sugar.
Two complementary strands - opposite directions (5’ to 3’).
Hydrogen bonding between nitrogenous complementary bases (A-T, C-G).
How is DNA packaged in…
Bacteria?
Archaea?
Eukarya?
B - single circular chromosomes, supercoiled.
A - single circular chromosome packaged around histones.
E - linear chromosomes (multiple) packaged around octamer histone proteins.
How does DNA replicate?
DNA replicates Semi-Conservative, meaning one strand from the original molecule is passed on to each offspring, and one new strand is synthesized.
Where does DNA replication begin in bacteria?
At the origin of replication (oriC). 245 bp in length. Can recognize sequences upstream (13bp sequence and 9-bp sequence) for the oriC, allows indication of where to replicate.
Bacterial DNA Replication: Initiation?
Initiation: dna-A protein interacts with 9-bp region of the oriC, then additional dna-A come binding to oriC forming a complex and DNA starts to unwind. dna-C delivers dna-B (helicase enzyme) which completely unwinds DNA and creates replication bubble.
Eukaryotic DNA Replication: Initiation?
Eukaryotic chromosomes have multiple origins of replication. The ORC (origin of replication complex) binds to ARS (autonomous replicating sequence). Proteins (Cdt1, Cdc6, MCM) are recruited. Then DNA unwinding occurs, with SSB proteins keeping DNA strands apart.
What do ARS stand for?
Autonomous replicating sequence. 10 - 100 kb apart.
What does MCM stand for?
Minichromosome Maintenance complex
Eukaryotic DNA Replication: Elongation?
DNA Polymerase III adds nucleotides to RNA primers (synthesized by primase). Continuous leading strand and discontinuous lagging strand. Okazaki fragments add fragments to join RNA primer and lagging strand. DNA primers are removed and replaced by DNA Polymerase I, sealed by DNA ligase joining phosphate to the sugar backbone.
Processivity
DNA polymerase ability to continue DNA synthesis without dissociation. Sliding DNA clamp protein (archaea/ eukaryotes, prokaryotes).
Archaea/ Bacteria DNA Replication: Termination (circular chromosomes)
Bidirectional replication starts at oriC, Tus proteins bind to ter sites (opposite of oriC) stopping elongation. Topoisomerase II is recruited, newly replicated circular molecules are disentangled –> 2 circular chromosomes.
Eukaryotic DNA Replication: Termination (linear chromosome)
RNA primer removed by Exonuclease, 5’ end cannot be extended by DNA polymerase. Telomerase extends 3’ end (on template DNA)
