Lecture 23 Flashcards
3 hypotheses for DNA replication
conservative
dispersive
semiconservative
conservative process
the original double stranded DNA is maintained after replicateion
the new double stranded is formed during replication process
dispersive process
each strand of the two daughter molecules would have some parts that are newly made, and some parts that are from the original
semiconservative process
the two old strands from the original DNA separate and each gain a new complementary strand during replication process
Meselson and Stahl
- tested the hypotheses of DNA replication
- used isotope of N which is heavier - note radioactive to label E.coli
- determined DNA replicates by a semiconservative process
Meselson’s and Stahl’s experiment
- grow E.coli in 15N media and sampleDNA
- transfer to 14N media
- replicate for 1 generation and sample DNA
- let replicate another generation and sample DNA
- let replicate a 3rd generation and sample DNA
- analyze with density gradient centrifugation
- 15N is heavier than 14N and will be closer to the bottom of the tube
- strands with both 15N and 14N will be in the middle of the tube
Taylor, Woods, and Hughes
- to show replication is semiconservative
- used autoradiography to examine chromosome during metaphase
- labels with 3H thymine or tritium
Taylor’s, Woods’s, and Hughes’s experiment
- initially not exposed
- then exposed to 3H for brief period in interphase of one cell cycle
- chromosome collected in metaphase of same cycle
- autoradiography performed to see where tritium incorporated
- both chromatids in metaphase labeled indicated they both contained some 3H
- then allowed replication for another cell division without tritium
- isolated chromosomes in metaphase of second cell division
- only one of the two chromatids labeled
- consistent with semiconservative
isotope of Taylor, Woods, and Hughes
tritium
3H
isotope of Meselson and Stahl
15N
Modes of semi-conservative replication
theta
rolling circle
linear
theta replication
common in bacteria and other circular DNA molecules
results in 2 circular molecules
bidirectional
rolling circle replication
used in conjugation when F factor is transferred to an F- cell
also used by viruses such as lambda bacteriophage
linear replication
used by eukaryotes
has multiple origins
proceeds bidirectionally
uses telomerase
bidirectional replication
replication can proceed in both directions form the origin
describe rolling circle replication
- one strand of DNA is nicked
- 5’ end lead the way out of the circle
- as the strand pulls out form the circle the inner strand rolls
- using the inner strand as a template, nucleotides are added to the 3’ end of the nicked strand
- replication also occurs using the nicked strand as a template
- the inner circle can continue rolling to allow many copies to be produced end to end
how are concatemers of lambda DNA produced?
by rolling circle replication
Theta Replication
- DNA template
- Breakage of strand?
- # of replicons
- uni or bidirectional
- products
- Circular
- No breakage
- 1 replicon
- either uni or bidirectional
- 2 circular molecule products
Rolling-circle Replication
- DNA template
- Breakage of strand?
- # of replicons
- uni or bidirectional
- products
- circular
- breakage
- 1 replicon
- unidirectional
- one circular molecule and one linear molecule that may circularize
Linear Replication
- DNA template
- Breakage of strand?
- # of replicons
- uni or bidirectional
- products
- linear
- no breakage
- many replicons
- bidirectional
- two linear molecule products
DNA polymerase III
- responsible for most DNA synthesis
- proofreads with3’-5’ exonuclease activity
DNA polymerase I
- removes and replaces primers
- uses 5’-3’ exonuclease activity to remove RNA primers
- proofreads with 3’-5’ exonuclease activity
What enzyme creates phosphodiester bonds?
DNA polymerase
In what organism was the research done to discover DNA polymerase?
ecoli
Which DNA polymerases function in replication
I and III
What is the main polymerase in replication
III
describe the reaction to form a phosphodiester bond
- catalyzed by DNA polymerase
- requires nucleoside triphosphates and 3’OH to add a nucleotide to
- the 2 distal phosphates are cleaved off
- the resulting phosphate on the 5’ end is added to the 3’ end of the existing strand of nucleic acid
can dna polymerase start a new strand from scratch?
no
Why does the lagging strand require discontinuous synthesis?
DNA polymerase can only add nucleotides 5’ to 3’ - must attach nucleotide to 3’OH
Okazaki fragments
- DNA constructed discontinuously on the lagging strand
- 1000-2000 nucleotide in prokaryotes
- 100-200 nucleotides in eukaryotes
Leading strand
continuous replication
3’ end exposed to have nucleotides added
replication moves in the direction of the direction of unwinding
Lagging strand
discontinuous replication
5’ end exposed, requires multiple initiation points
forms Okasaki fragments
DNA must be produced in a ____ direction.
5’ to 3’
lagging strand in rolling-circle replication
the strand that pulls out of the conjugation tube - 5’ end leads out
leading strand in rolling-circle replication
the end left behind with a 3’ end exposed
leading strand in linear replication
- origin is at the 3’ end allowing nucleotides to be added 5’ to 3’ in an antiparallel manner
lagging strand in linear replication
origin is at the 5’ end
must be added in segments
initiator protein in e.col
DnaA
binds to origin of replication causing local unwinding and a short stretch of single-stranded DNA
helicase in e.coli
attaches at replication fork and moves into the fork breaking H-bonds as the replication fork moves along the DNA
SSB in e.coli
binds to single stranded DNA to stabilize it and prevent hairpin formation
prevents the DNA strands from reannealing
gyrase in e.coli
a topoisomerase
relieves supercoiling ahead of replication fork
how does gyrase work?
relieves the tension ahead of the replication fork by binding to the phosphate of the DNA causing a break in the phosphodiester bond
the two ends of the DNA can now rotate relative to each other and then reseal
How does primase help initiate elongation in replication in ecoli?
can start strands from scratch
joins nucleotides of RNA together with phosphodiester bonds forming an RNA primer as the DNA opens up
contains a 3’OH end
DNA polymerase III can add nucleotides to the 3’ end of the primer
the primer will eventually be removed and replaced with DNA
primase in e.coli
RNA polymerase
forms RNA primers
Describe elongation of replication in the leading strand in ecoli
- primase begin RNA primer
- DNA polymerase III adds nucleotides to the 3’OH of the primer
- addition of bases continues in the direction of unwinding
Describe elongation of replication in the lagging strand in ecoli
- primase forms multiple RNA primers along the strand
- nucleotides are added to the 3’OH in the opposite direction of unwinding
- continues from one primer until it reaches the other
- DNA polymerase I then removes the primer and fills the gap with nucleotide
- the last sugar phosphate bond is filled by DNA ligase
primosome in ecoli
complex of helicase and primase
gap in ecoli
missing nucleotide
nick in ecoli
missing sugar phosphate bond
DNA polymerase I in ecoli
removes RNA primer and fills in gap
DNA ligase in ecoli
seals nick
DNA polymerase III in ecoli
adds nucleotides to the RNA primer
Main differences between prokaryotic and eukaryotic DNA replication
In eukaryotes…
- many origins of replication exist
- origin must be licensed for replication to occur
- linear chromosomes rather than circular
- lots of different DNA polymerases with various roles
- replication of telomeres
- nucleosome assembly immediately follows replication
Licensing of DNA Replication
- makes sure each piece of each chromosome is replicated once and only once per cell division in eukaryotic cells
replication licensing factor
attaches to each origin of replication early in cell cycle
activated just after mitosis and before replication starts
is removed as replication proceeds from the origin
Replication in a eukaryote will only start…
at licensed origins
Problem of telomeres in eukaryotes
- the gap at the 5’ end of new strand cannot be filled once the RNA primer is removed because there is no 3’OH available at the end of the chromosome
- leaves gap of 70-100 nucleotides each replication
solution to the problem of telomeres in eukaryotes
telomerase
telomerase
ribonucleoprotein
contains RNA which it uses to make several repeats of DNA to fill the gap
ribonucleoprotein
a protein that complexes with RNA
describe action of telomerase
- uses reverse transcription
- DNA is added
- telomerase moves along the DNA
- eventually telomerase is removed
- DNA polymerase fills in the rest of the other strand
