Meselson-Stahl experiment - supported Watson/Crick’s theories on DNA replication
- DNA replication theories
- semiconservative replication - each strand of DNA duplex used when forming new DNA
- conservative replication - original DNA duplex remains intact, new DNA has only new molecules
- dispersive replication - original DNA gets scattered in new DNA, which contains new/old molecules on each strand
- bacteria w/ heavier nitrogen (15N) isotope in DNA grown and then transferred to bacteria w/ lighter nitrogen isotope (14N)
- centrifuge used to determine density of DNA after replication
- results showed that DNA replicates in a semiconservative way
replication process - must be fast/accurate
- starts at origin, goes bidirectionally towards the terminus
- replicon - functional unit containing chromosome and origin
- polymerase - enzyme that synthesizes nucleic acids
- 3 main ones: pol I, pol II, pol III
- DNA pol II used mainly for DNA repair
- DNA pol III - made up of alpha subunit (main catalytic part) and beta subunit (forms ring around template, acting as sliding clamp)
- primer - short stretch of DNA/RNA nucleotides hydrogen-bonded to the complementary strand
- cannot start synthesis of DNA w/o primer
- endonucleases - cuts DNA internally
- exonucleases - chews away at end of DNA; helps proofread
- used by DNA pol I to remove the primers after replication
- leading strand - can be replicated as 1 continuous strand, uses 1 primer
- lagging strand - replicated only in short stretches (Okazaki fragments), uses multiple primers
- DNA primase - synthesizes short RNA primer
- RNA polymerases don’t need primers to start
- DNA helicase - enzyme that unwinds/opens the DNA strands
- DNA gyrase - form of topoisomerase that takes away the torsional strain (coiling up of strands)
- single-strand binding protein (ssb) - covers the hydrophobic single, unwound DNA strands
- DNA ligase - creates phosphodiester bond to join the Okazaki fragments
- replication fork - site where DNA strands open and replication occurs
- replisome - replication organelle, assembly of proteins
- primosome - made up of primase/helicase and other proteins
- 2 DNA pol III, 1 for each strand
- both pol III move in the same direction, but 1 of the strands looped
stages of replication -
- initiation - occurs at the origin (OriC)
- initiator protein recognizes specific sites within the OriC
- opens up helix at A-T rich region (very few triple bonds)
- primosome assembled onto strands
- 2 replication forks form as replication goes bidirectionally
- elongation - takes up most of the time during replication
- pol III add new nucleotides to the template strand
- more complicated process on lagging strand than on leading strand
- termination - termination site located opposite the origin on circular chromosome
- DNA gyrase keeps new DNA molecules from intertwining
eukaryotic DNA replication - main difference in amount of DNA reproduced
- uses multiple origins for replication
- more origins/replicons formed when divisions need to be rapid
- PCNA (proliferating cell nuclear antigen) - replaces beta subunit in eukaryotes
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