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Lecture 6

BIL 255 Lecture Notes - Lecture 6: Deamination, Okazaki Fragments, Homologous Recombination


Department
Biology
Course Code
BIL 255
Professor
Diresta Dan
Lecture
6

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Ch. 5 DNA & Chromosomes
Griffith cells contain some component of that can be transferred to a new population of cells
and permanently cause changes in the new cells
Hershey and Chase obtained definitive results demonstrating that DNA is the genetic
molecule
Avery, Macleod, & McCarty identified the class of biological molecule that carries heritable
iforatio, trasforig priiple
Ch. 6 DNA Replication, Repair, & Recombination
DNA Replication:
DNA acts as a template for its own duplication
Added 5’3’ diretio
Read 3’5’ diretio
Meselson-Stahl Experiment
Semiconservative
o b/c each daughter DNA double helix is composed of one conserved strand and
one newly synthesized
Separated light and heavy DNA by density
Incorporation of new nucleotides to growing chain
Energy from incoming nucleotide
Two step reaction:
o Base pairing
o Covalent bond
Chai gros 5’3’ diretio
o Base is added to the 3’ aro o the preious ase
10,000 replication origins in human vs 1 replication of origin in bacteria
What would happen if ddCTP were added with dCTP?
ddCTP would randomly bind and stop the synthesis
FUNCTIONS of ENZYMES in DNA REPLICATION:
DNA Helicase unwinds DNA double helix
DNA Polymerase adds uleotides i the 5’ to 3’ diretio; also proofreads ad error
corrects; many different types
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DNA clamp prevents DNA polymerase III from dissociation from the DNA parent strand
SSB Proteins bind to ssDNA and prevent the DNA double helix from re-annealing
Topoisomerase relaxes DNA from super-coiled state; relieves strain
DNA Ligase re-anneals the semi conservative strands and joins Okazaki Fragments of
the lagging strand
Primase provides a starting point of RNA (or DNA) for DNA polymerase to being
synthesis of the new DNA strand
Telomerase lengthens telomeric DNA by adding repetitive nucleotide sequences to the
ends of eukaryotic chromosomes
o Allows germ cells and stem cells to avoid the Hayflick limit on cell division
Multi-enzyme complex
DNA Template
dNTP’s
Enzymes
o DNA dependent DNA polymerase
o Primase (RNA polymerase)
o Helicase
o Topoisomerase
o SSB protein
o Clamp
Primer! (RNA polymerases do not need it
DNA dependent DNA polymerase
Many sub-types
1000 bp/sec
Two catalytic sites
o Polymerizing site (P site)
1 error every 1x105 bases
doule hek ehais
base pairing leads to conformational change
conformational change leads to bond formation
o Editing site (E site)
“thesis 5’3’
Contains separate sites for DNA synthesis and proofreading
Proofreading by DNA Polymerase
Exonucleolytic proofreading 3’5’ eoulease
o In a diff. domain of the DNA polymerase
o Occurs concomitantly with strand elongation
o If a iorret ase is added, it is upaired efore reoal
Stalling mechanism in P brings misincorporated nucleotide to E site
Decreases error rate 100 times to 1 error every 1x107 bases
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Wh does the hai gro 5’3’?
Proofreading mechanisms would terminate chain elongation
Backstitching mechanism
Must oe 3’5’ eause DNA polerase atalzes the joiig of a uleotide to groig
DNA strand by hydrolysis of pyrophosphate (PPi) to inorganic phosphate (Pi) + Pi
Asymmetrical replication forks
Leading strand: continuous synthesis
Lagging strand: discontinuous synthesis
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