Lecture 3 Flashcards
Why does DNA need to avoid mutations?
Germ cells must avoid mutation to maintain the species (aka pass on high quality genes)
Somatic cells must avoid mutation to avoid uncontrolled proliferation (cancer)
How does the DNA polymerase enzyme conduct DNA replication?
DNA polymerase replicates DNA in a “template directed” fashion that is complementary to the template strand
What level of fidelity does DNA replication occur at?
high fidelity, out of neccesity
What are the requirements for DNA polymerase to conduct DNA replication?
Separation of the DNA strands
dATP, dGTP, dCTP and dTTP (these represent the different base pairs that will be used to construct DNA)
A primer with a free 3’ -OH in order to begin replication
The replication fork is _______ and both strands are replicated simultaneously in the _______direction
asymmetric ; 5’ to 3’
List the proteins involved in DNA replication
DNA Primase RNAseH DNA Ligase DNA Helicase ssDNA binding proteins Sliding clamp Clamp loader
What does RNAseH do?
recognizes RNA primer and DNA hybrids and replaces RNA with DNA
What do ssDNA binding proteins do?
bind to the cooperatively exposed single strand DNA strands
Stabilizes single DNA strands
Prevents “hairpin” formation
Define the Sliding clamp protein
keeps the DNA polymerase “clamped” on the strand as it moves along
Releases when double stranded DNA is encountered
Define the Clamp Loader protein
hydrolyzes ATP to load the clamp (and DNA polymerase) onto primer regions of the template junction
works in conjunction with the sliding clamp
Does the Clamp loader protein associate more with the leading or lagging strand?
Hangs around the lagging strand more closely because it needs to help load the clamp onto the strand between each fragment (doesn’t need to do this to leading strand)
Describe the mechanism of Type I Topoisomerase
breaks a single strand of DNA and uses the other phosphodiester bond as a “swivel point”
Thermodynamically favorable process that doesn’t require the addition of energy
(Energy is supplied by the phosphotyrosine linkage)
Describe the mechanism of Type II Topoisomerase
breaks both strands of the DNA, lets the strands unwind, and then reseals the bond
Requires energy from ATP
Often used to separate 2 intertwined circular strands of DNA (usually in bacteria)
Describe the proofreading function of DNA polymerase
a conformational change that occurs during the act of adding a single nucleotide (the green hand “squeezing” down on the new nucleotide)
Any replication errors disrupt the conformational change and be ID’ed as an error
Describe Exonucleolytic Proofreading function
conducted by the exonuclease enzyme that works in the 3’ to 5’ direction (opposite of DNA polymerase) and will clip off the unpaired residues at the 3’ primer terminus
