lect 15: DNA replication Flashcards
what are the learning objectives of this lecture?
-explain the concept of semi-conservative DNA replication
-outline the process of DNA replication in bacteria and eukaryotic cells
-distinguish the functions of the different DNA polymerases
-explain how the end replication problem is solved
-list the general mechanisms of DNA repair
DNA: a __________ for its own replication
each strand acts as a template independently
-fast: synthesis is very fast for strands (1000 nucleotides/second)
-accurate: if mistakes happen (rare), so proof read and repair mechanisms
what is it that enables it to act so nicely?
-complementary base pairing (good at communicating)
what are the proposed mechanisms of DNA replication?
conservative
-DNA parent molecules is copied as a whole
semi-conservative
-2 strand separate each strand serves as a template for a new strand
-each has 1 strand from parent
dispersive
-parents get cut up and replicated and stitched together (alternate)
figured out by putting cesium chloride and centrifuged to see weight of the molecule (at different locations due to weight)
how did we determine the mechanism of DNA replication?
took bacteria (2 types) and grew in different mediums (different nitrogen isotopes, which have different weights)
-separated bacteria in 15N=lower (heavier), and 14N=higher (lighter)
how did we get to the conclusion of proposed DNA mechanism?
isolated bacteria in heavy, placed this heavy bacteria into a light medium
-then isolated and centrifuged
-if it was conservative, it should have 2 distinct bands (light and heavy)
-but results were in the middle
what is the mechanism of DNA replication?
-double-stranded DNA molecule is copied to produce two identical DNA molecules
-each DNA strand can act as template
semi-conservative
-each daughter duplex contains one strand from parent
what are the steps of DNA replication?
- identification of the origin of replication (ORI)
-control where we begin - unwinding of the DNA
-separate double helix so we can use as templates and add replication machinery - formation of the replication fork
-expands from the origin of replication - actual DNA synthesis
-creates phosphodiester bonds between nucleotides - termination of DNA replication
what is DNA replication in bacteria?
replication starts at a single origin site
-OriC is a specific DNA sequence in E. coli
-where proteins bind to initiate DNA replication
-recognized by replication machinery
replication forks
-parental double strand DNA helix unwound
-nucleotides incorporated into newly synthesized strands
-end where DNA is coming apart and moving forward
replication is bidirectional
-start in places in different directions and meet in the middle
what is the problem with unwinding the duplex and separating the strands?
-as DNA strands are separated, the unseparated portions become more tightly wound
-this makes them harder to separate
what is graph of the problem?
how does the cell deal with this unwinding problem?
topoisomerases
-two different types
what are the types of topoisomerases?
topo I
-cuts one of the strands of DNA and unwraps it and seals it back together
topo II
-cuts both DNA strands, causes unwrapping of the coiling (relieves torsial strain)
-requires ATP
what is DNA gyrase?
its a type II topoisomerase (in E. coli)
-travels in front of replication fork, creating double stands DNA breaks and sealing them
-ATP-dependent
what are DNA polymerases?
what is the meaning of semi-discontinuous replication?
-simultaneous synthesis of daughter strands
-DNA polymerase travels along template in 3’ to 5’ direction
-leading strand: synthesized continously
-lagging strand: synthesized discontinuously
what is the function of DNA polymerase III and primase?
what is the DNA polymerase I and DNA ligase?
DNA polymerase I
-removes RNA primers
-fills gaps between okazaki fragments
strands sealed by DNA ligase
what is the machinery operating at replication form in bacterial cells?
what is the structure and function of DNA polymerase III?
what are the three distinct activities of DNA polymerase I?
5’ to 3’ polymerase (synthesize in between okazaki fragments
5’ to 3’ exonucleases (remove/cut nucleotides)
-primer removal
-fix nicks in DNA
3’ to 5’ exonuclease
-corrects mismatches (“proof reading”)
what is the graph of DNA polymerase I activity?
what is the initiation of replication in eukaryotic cells?
multiple origin sites
-replication forks progress in both directions
-human cell: 10,000-100,000 origins of replication
-control points exist to ensure DNA only replicated once per cell cycle (very precise control)
what is the table of the proteins required for replication?
what is the eukaryotic replication fork?
what is the end replication problem in eukaryotes?
what is the solution to the end replication problem in eukaryotes?
telomerase
-extends template strand in 3’ to 5’ direction
-adds repeated telomere sequences so we get back filling in of complimentary DNA strand for that region (adds length)
what are the differences in DNA replication between prokaryotes and eukaryotes?
DNA is the most __________
DNA is most senstitive and susceptible to damage
-DNA repair is essential for cell survival: the most susceptible biological polymer
several mechanisms to repair genetic damage:
-nucleotide excision repair
-base excision repair
-mismatch repair
what are the mechanisms to repair genetic damage?
nucleotide excision repair (remove entire nucleotide)
-cut and patch mechanism
-removes pyrimidine dimers, nucleotides with various chemical groups
base excision repair
-removes base altered by reactive chemicals in the diet/produced by metabolism
mismatch repair
-mismatched bases incorporated by DNA polymerase that escape the enzyme’s proofreading exonuclease
