Lecture 7 Flashcards
Two of the most important biological investigations of the twentieth century were to
1) find what is the genetic material of life
2) elucidate the structure of that material
the genetic material of organisms was unknown. Possible candidates included
polysaccharides, DNA, lipids, proteins, etc.
many thought that ___ were the most likely candidate for the genetic material of organisms given ___
proteins
DNA –> simple repetitive molecule
proteins –> thousands of various forms.
In a series of famous experiments by ____ the case was settled, DNA was the hereditary molecule
1) Avery, MacLeod, and McCarty (1940’s)
2) Hershey and Chase (1951/1952)
the famous experiments that showed DNA was the hereditary molecule
infect mouse with virulent S strain bacteria (combined with a living non-virulent R strain)
only when the DNA of the virulent strain is destroyed that the mouse lives
One key to that lead to the discovery of the structure of DNA came from ____
who ___
Rosalind Franklin
- stretched vicious fluids containing DNA
- and projected x-rays through the strands
- and used photographic plates to gather the refracted x-rays.
Depending on the preparation method (e.g. pH, and water content) she found differing patterns (for DNA structure, A-DNA and B-DNA)
She was methodical in her approach beginning work on the ___
A-form (A-DNA)
but the B-DNA form represented the typical biological shape of DNA. this form was worked on by J Watson and F Crick
Professor Franklin was a ___
Her primary school was unusual because __
she worked in the ___ field
she died in ___ at the age of _____
intellectual power recognized even in childhood
it prepared girls for careers rather than being a typical house wife
in many fields
1958 at the age of 38.
June (Broomhead) Lindsey was a ___
awarded her first degree in 1944 (doctorate in 1950), and then joined the ___ where she ___
gifted researcher
Cavendish lab at Cambridge
discovered base shapes and H bonding in DNA
____ proposed that “nucleobases” are bound to each other via hydrogen bonds
June (Broomhead) Lindsey
____ directly used ____ work to clarify the structure of DNA. However, they did not cite her papers or give her any credit.
Watson and Crick (“discovered” the structure of DNA in 1953)
Lindsey’s
DNA structure is a ___
with ___ as backbone
double helix with
phosphates & pentose sugars –> backbone
and nucleotides in the middle.
Sugar in DNA ___
Phosphates in DNA ___
lacks a typical oxygen and thus “de-oxy” ribose nucleic acid;
have a negative charge making DNA slightly acidic (thus a nucleic “acid”).
The nucleotides have a specific complimentary pairing of
one purine (two ringed molecule) with one pyrimidine (one ring), A to T and G to C.
Purine vs Pyrimidine
Purine - A & G
Pryimidine - T & C
(AT, GC)
ratio of AT/CG
is variable & differs significantly by the taxonomic group (organism AND part of DNA loking at –> AT/CG rich regions)
____facilitates the macro structure of DNA.
The specific complimentary bonding of the nucleotides
____ makes the DNA much more stable and thus less likely to degrade.
very tight spacing of the molecule, excludes H2O
stacked in middle –> single/double allows adjacent (above and below) close stacking –> excludes H2O
the shape of the molecule produces a ”major” grove that is larger, and a smaller “minor” grove.
these spaces are important ___
for proper protein (structural and regulatory) binding to the helix.
chromatin
DNA + proteins (e.g. histones)
actual/typical structure of DNA
tends to be condensed via wrapping around proteins (e.g. histones) into chromatin. “middle ground” - not completely uncondensed or fully condensed in cell devision
The basic histone spool is a
octamer of 8 subunits (2 copies of 4 proteins)
____ facilitate DNA spooling.
are additional histones (e.g. H1)
The regulation of chromatin (histone) binding is one key factor in
the regulation of DNA replication and transcription.
Chromatin remodeling
chromatin (histone) binding
Histones are generally moved
down the DNA allowing specific sequences to be transcribed
histone tails are modified (acetylation, de-acetylation) to
release or bind to DNA
Pattern of DNA replication is
semi conservative
famous experiment by Meselson and Stahl using ___ established that ____
labeled DNA (containing heavy N) for the initial DNA strands, and unlabeled (light) DNA (nucleotides) for subsequent replication
replication was (and is) in fact semiconservative
potential models for DNA replication
Semiconservative - 1 old 1 new
Conservative - all old or all new
Dispersive - altering segments of old and new
Eukaryotic knowledge lags behind Prokaryotic because of
its increased size and complexity
The biochemical machinery of replication
replisome
DNA replication is termed ____________ because of the lagging strand ____
semi discontinuous
Ligase connects the fragments on the lagging strands
One physical problem with splitting the strands of DNA is that this puts
tension on the adjacent portions of the DNA
These “overwound” regions form loops and super coils that the helicase could not move
Supercoiling bacterial chromosomes vs eukaryotes
Less prevalent un eukaryotes
The cell solves the supercoiling issue by
(Prokaryotic)
periodically cutting the DNA with DNA Gyrase.
releases tension in the area and allows the coils to unwind.
DNA Gyrase does ____
Type of ___
snips both DNA strands, so they can uncoil and then glues/sticks them back together
type of topoisomerase
(Prokaryotic)
Bacterial DNA replication is initiated at a single location known as the
origin (oriC)
region - 245 bases, five copies of 9 bases (DnaA boxes) that facilitate protein binding.
“boxes” are adjacent to an A-T rich region.
DnaA Boxes
protein factors bind to them, stabilize and break H bonds in AT rich regions
also help prevent the DNA strands from re joining
factors regulate DNA replication and the cell cycle in bacteria
(Prokaryotic)
periods in transcription and translation
1) initiation
2) elongation
3) termination
first step of the bacterial DNA replication process is
1) DnaA proteins bind to DnaA boxes.
2) DnaA proteins help stabilize the area and break H bonds in AT rich regions (and prevent rejoining of DNA)
3) two helicases (hexamer) to bind on the opposing strands - continues opening the DNA
4) larger opening - recruits other parts of replisome
Why break AT rich region first?
CG region would required more energy use
DNA Polymerase uses what energy
energy stored in the triphosphate of the incoming nucleotide to bond to the adjacent sugar
two phosphates are byproducts of rxn
principle Prokaryotic polymerases
- DNA polymerase III - main work of replication on both strands
- polymerase I - closes up fragments on the lagging strand
- polymerase II - proofreading functions, and helps if poly III becomes stalled.
(are also 2 others with limited functions)
Eukaryotes DNA polymerases
many more DNA polymerases in Eukaryotes than Prokaryotes
two most important Eukaryotic DNA polymerases are
1) epsilon - leading strand synthesis
2) delta - lagging strand
BOTH - fast and accurate
issues polymerases often encounter
be gaps, proteins in the way, and mismatched bases
____ does not function efficiently in trouble areas of the DNA
(Eukaryotic)
Epsilon
can stall and disassociate from DNA
other polymerases termed _______ can be substituted when issues arise in eukaryotic DNA sysnthis
translesion DNA Polymerases
- can pass by or work through region to continue replication
- not as fast or accurate
structure of DNA polymerase is often described as
a hand with “palm, fingers, and thumb”.
hand - polymerase wrapping around the DNA to be replicated
thumb - appears to open and close around the DNA
(note spot for polymerase activity and exonuclease activity (proof reading))
DNA polymerase III (Prokaryotes) requires ____
This is done by a set of proteins called the ____
a small section of RNA “primer” (about 11 nucleotides) to be begin replication.
primeosome, it occurs once on the leading strand and for every fragment on the lagging strand.
only the leading strand can be complimented continuously because
Polymerase only adds nucleotides on the 3’ end of the growing strand
The lagging strand, must be added to discontinuously in ____ fragments.
_____ fills in the gaps on the lagging strand (3) and _______ connects the fragments (4).
(Prokaryotic)
~1,000 to 2,000 b.p. “Okazaki”
DNA polymerase I
DNA ligase
____ protein that keeps the polymerase attached to the strands
(Prokaryotic)
B clamp
without the clamp the polymerase would quickly detach from the strands
_______ stabilize the open areas until replication has been achieved
(Prokaryotic)
single strand binding proteins
SSBP
differences between the replisomes of Prokaryotes vs. Eukaryotes may be sites for novel
anti-bacterial antibiotics
Eukaryotic genomes use ____origins of replication
many, sometimes tens of thousands
Each site has its own set of DNA replication enzymes
Eukaryotic DNA replication Initiation
ORC–> Cdc –>Cdt w/helicase
- Occurs during the S phase of the cell cycle
- Origin of replication complex (ORC) recognizes origin region (AT rich) and 11pb sequence
- There are many origins of replication, each site has its own set of DNA replication enzymes
- Binding of ORC stabilized by cell division control factors (Cdc’s)
- Cdt (control factor) helps recruit helicase
- Helicase binds and ORC is released
- Helicase unwinds DNA and stabilizes the area so DNA poly complex can bind
Prokaryotes origin of replication
only one
that region is highly conserved and easily identified.
Rates of DNA replication for a single site can be as high as
1000 bases per second.
One of the reasons for this accuracy is that polymerases have proof reading ability.
Proofing often takes place by:
1) Mismatch recognition
2) exonuclease removal by polymerase
(also proofing mechanisms based on recognition and endonuclease (not exo) activity.)
3) normal extension adding correct base
the lagging strand (in eukaryotes) needs
primers that would extend out over the end of the sequence.
Because primers cant be placed beyond the end of the sequence there is a overhang that does not get complemented.
This overhang generally gets degraded and thus the DNA gets shortened with every cell cycle.
Cells such as ____ have a mechanism such that their chromosome ends are not shorted every generation
this mechanism is ___
germ line cells, stem cells, and a few others
the enzyme complex telomerase, that helps to produce telomeres.
Telomerase enzyme complex– has its own ____ allows ____
____ fill in gap – still ___
own small sequence of RNA, that acts as primer,
allows lagging to be lengthened (adds 6-9 nucleotide G/C rich region)
Primase and DNA polymerases fill in gap
still small single strand but not as bad
Telomere Structure
single strand can tuck back into DNA making telomeric loop (t-loop)
hundreds of repeats that are then bound in a protein “cap” with the total structure being called the telomeric loop (t-loop).
why are telomeres not a “fountain of youth”.
because in normal cell lines telomere shorting is a normal timer for cell death, if do not stop normal cell death get cancer
Werner syndrome
causes rapid aging
is associated with rapid telomere decay.
