Lect. 3 - Replication, repair and recombination 1 Flashcards
mutation rate in human germ line
one nucleotide change per 10^8 nucleotides per generation
mutation rate in e.coli
one nucleotide change per 10^10 nucleotides per cell generation
What is a critical need for multicellular organisms?
HIGH FIDELITY REPLICATION; Germ cells have to have low mutation rates to maintain the species and somatic cells need low mutation rate to avoid uncontrolled proliferation/cancer
DNA polymerase
synthesizes DNA by catalyzing the following rxn: (DNA)n residues + dNTP –> (DNA)n+1 residues + P2O7
What does DNA polymerase require to begin?
a primer w/ a free 3’ -OH to begin
What direction can the DNA polymerase synthesize in?
5’ to 3’ direction
How often does DNA polymerase make a mistake?
1 out of every 10^9 nucleotides copied, thanks to proofreading.
What step is first just before a new nucleotide is added?
enzyme must tighten its “fingers” around the active site, which is easier if the correct base is in place
Exonucleolytic proofreading
DNA polymerase requires a perfectly paired 3’ terminus’ 3’ to 5’ exonucleuase clips off unpaired residues at 3’ primer terminus
When does exonucleolytic proofreading take place?
immediately after incorrect bases is added
Why 5’ to 3’?
Allows for efficient error correction; that direction is energetically favorable to remove a new chain and start over.
Lagging strand synthesis
replicated through backstitching process
what does DNA primase do?
synthesizes an 10nt long RNA primer to prime DNA synthesis b/c if DNA polymerase initiated it it would increase mutation rate
RNA primer
erased by RNAseH (recognizes RNA/DNA hybrids) and replaced w/ DNA;
What joins the ends after DNA replaces the RNA?
DNA ligase
DNA helicase
unwinds DNA; protein w/ 6 identical subunits that binds and hydrolyzes ATP; this confomational change that propels it like a rotary engine along single stranded DNA, passing it through a center hole.
Capable of prying apart the helix at rates of 1000 nucleotide pairs/sec
Single-stranded DNA binding proteins
bind tightly and cooperatively to exposed SS DNA; it is less energetically favorable so will want to try and make a double strand if possible (or bind to itself = hairpin)
what 3 things do the single-stranded DNA binding proteins do?
- help stabilize unwound DNA
- prevent formation of hairpins
- DNA bases remain exposed
Sliding clamp
keeps DNA polymerase on DNA when moving; releases when double stranded DNA is encountered (allows long stretches of DNA rep. to occur)
clamp loader
hydrolyzes ATP as it loads the clamp onto a primer-template junction
On the leading strand, what does the clamp do?
remains associated to DNA polymerase for long stretches
On the lagging strand, what does the clamp loader do?
stays close so it can assemble a new clamp at start of each new Okazaki fragment
Mismatch Repair
removes (almost all) errors missed by proofreading by detecting distortion caused by mispairing
Which strand is correct?
the methylated strand is correct in e.coli and in humans, depends on single strand breaks - present on lagging strand before Okazaki fragments are ligated, leading strand not known.
