Transcript DNA Replication

Molecular Genetics
DNA Structure and Replication
Chapter 11
History of Molecular Genetics
* In the pasts, scientists argued about how
genetic information was passed down and
influcenced traits.
* Some scientists believed in "blending" that the traits of two parents are mixed.
* This doesn't work when a white flower
and a
purple flowered plant have a
white flowered
offspring.
* Some scientists also believed that
proteins were responsible for passing on
traits, not DNA. They thought this
because DNA molecules are so simple
compared to proteins.
Current Thinking and the Experiments that Led to It
Griffith & Avery
* Took pathogenic bacteria (cause disease) and killed
them with heat.
* Mixed the dead bacteria with harmless bacteria.
* When they injected the mix into mice, it killed them.
* The harmless bacteria must have taken up something from the dead, harmful
bacteria that transformed the harmless bacteria
* It was the DNA!
Current Thinking and the Experiments that Led to It
Avery, MacLeod, and McCarty
* Tested what was being
transferred between the
bacteria...was it DNA? RNA?
Proteins?
* Killed virulent bacteria
* Subjected a sample to
RNAase, one to
Protease, one to DNAase
* The samples treated with
RNAase and Protease
were still able to
transform harmless
bacteria but DNAase
treated sample could not
* DNA must be the agent of
transformation
Current Thinking and the Experiments that Led to It
Hershey & Chase
* Radioactively labeled DNA and proteins in viruses.
* They allowed the viruses to infect cells
* Waited to see whether it was the DNA or the protein that entered
the cells to infect them.
* Found DNA to be radioactive and therefore the genetic material
Current Thinking and the Experiments that Led to It
Chargaff's Rule
* Identified pairing rule of bases
* Adenine = Thymine (2 H bonds)
* Guanine = Cytosine (3 H bonds)
Rosalind Franklin
* took the first picture of a DNA molecule using
a technique called x-ray crystallography
Watson & Crick
* used Franklin's picture and Chargaff’s ratios
and cutouts to determine DNA structure
DNA Structure video
https://www.youtube.com/watch?v=C1CRrtkWwu0
DNA Structure
Nucleotide - monomer of nucleic acids
* DNA Nucleotide Structure - 3 Parts
1. Sugar (deoxyribose)
2. Phosphate group
3. Nitrogenous Base - 4 Kinds
1. Adenine
2. Guanine
3. Cytosine
4. Thymine
* Purines and Pyrimidines are ring structures
associated with the Nitrogenous base
* Purines - 2 rings
*EX: A and G
* Pyrimidines - 1 rings
*EX: T and C
DNA Structure
DNA is a molecule made of 2 strands of nucleotides
* Individual strands are made by connecting sugars and
phosphates of nucleotides with a phosphodiester bond
(type of covalent)
* Forms sugar - phosphate backbone
* The two strands are connected by hydrogen bonds
between the bases
* The two strands are wound together into a double
helix.
DNA Structure
Directionality of DNA strands
* 3' end - end of each strand
that has the hydroxyl
group off the 3rd
carbon of the sugar
molecule
* 5' end - the other end which
has a Phosphate
group off the 5th
carbon of the sugar
molecule
* Anti-parallel- The 2 DNA
strands run in opposite
directions to each
other
DNA Organization
DNA Organization
* Chromosome - DNA wrapped around histone proteins (before cell division)
* Chromatin - loose, uncoiled DNA (during Interphase)
* Euchromatin - areas in chromosomes that are loosely opened so it can be
transcribed
* Heterochromatin - areas in chromosomes that are compacted and generally not
transcribed
* Gene - A segment of DNA that codes for a specific trait
* Telomeres - repeated nucleotide sequences at the ends of chromosomes
* they do NOT code for proteins
* TTAGGG in humans -- it is repeated about 2500 times
* 50 -200 base pairs of telomeric DNA are lost after every cell division
* Cells die after 20 - 30 divisions because telomeres are gone
* Telomerase - some cells (bone marrow, stem cells, etc...) contain this enzyme which
regenerates telomeres.
* Present in 90% of cancer cells as well
DNA Replication
* Occurs during Synthesis phase of a cell's life
* This is done to prepare for cell division so
that each daughter cell is identical and
contains all of the necessary chromosomes
* Occurs during 2 major stages
1. Splitting the DNA
2. Building the new strands
DNA Replication
1. Splitting the DNA
* Helicase - an enzyme that breaks the
hydrogen bonds holding the
two strands of DNA together.
*starts at TATA boxes
* Replication fork - point where the
DNA opens
* Single strand binding proteins - hold
the strands apart during
replication
DNA replication simple
http://www.dnatube.com/video/365/DN
A-Replication
DNA Replication
2. Building the new strands
* Primase - enzyme that lays down a short RNA primer (necessary for DNA
polymerase to attach. This acts as a starting spot.
* DNA polymerase – attaches new nucleotides to the growing strand. DNA can only
add new nucleotide in the 3’ direction
DNA Replication
* Leading and lagging strands
* DNA polymerase can only add nucleotides in the 5' - 3' direction (adding to the 3’ end)
* Leading strand - runs 5' - 3'. Can be built continuously.
* DNA Polymerase III – the enzyme that adds new nucleotides to build the growing
LEADING strands.
DNA Replication
* Lagging strand - runs 3' to 5'. Must be built in chunks called Okazaki fragments
As helicase unzips the DNA strands, RNA primase lays down an RNA primer.
DNA polymerase II can now attach to the 3’ end of the RNA primer and adds
DNA nucleotides in the 3’ direction. The RNA primer is later replaced with DNA
nucleotides by DNA polymerase I.
* Ligase - enzyme that stitches fragments of sugar-phosphate covalent bonds together
DNA Replication
Extra phosphates on each
nucleotide provide the
energy for the process.
Called "active monomers" or
"phosphorylated nucleotides"
DNA replication detailed summary
http://www.youtube.com/watch?v=vNXFk_d6y80
DNA Replication video detailed!
http://www.dnatube.com/video/4197/
Process-of-DNA-Replication
DNA replication real time video
http://www.hhmi.org/biointeractive/dnareplication-advanced-detail
DNA Replication
DNA Replication
• DNA Replication is often referred to as
semi – conservative
* Semi-conservative - Each new molecule
of DNA is composed of one old
(parent) strand one new
(daughter) strand
Replication and Telomeres
• Telomere- ends of linear chromosomes with repetitive DNA
sequence: TTAGGG
• Telomeres allow cells to distinguish chromosome ends from broken
DNA and protect the DNA during cell division
• Telomeres help to delay cell death
• Telomeres shorten with each cell division because DNA polymerase
leaves 50-200 bp unreplicated on the lagging strand.
• Cellular senescence is triggered when telomeres are 4-6 kb
DNA Replication and Telomeres
Telomerase- enzymes that adds telomeres to end of DNA strand.
Only found in embryonic and stem cells (and cancer cells)
DNA Repair
DNA suffers from environmental stresses, radiation, UV light, and carcinogens,
which modify the DNA. In addition, internal errors formed during replication
must be corrected
* If any mistakes (mutations) are found, nucleases, polymerase
and ligase will fix it
* Nucleases - enzymes that double check that no mistakes
were made
DNA Replication - Overview
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Molecular Genetics
DNA  RNA  Protein
Chapter 12
* DNA "makes us who we are"
by coding for proteins
DNA & Proteins
* These proteins are responsible for our various
phenotypes
* EX: DNA determines the color of the protein
pigment made in your eyes and the amount
of melanin pigment made in your skin that
gives you your skin color.
* Protein Synthesis - process by which a protein is
made from the instructions contained in DNA
* Requires help from another nucleic acid called
RNA
RNA Structure & Function
* RNA = Ribonucleic Acid
* Differences between RNA and DNA
1. RNA is single stranded
2. RNA has Uracil instead of Thymine
3. RNA has ribose, not deoxyribose
* Three kinds of RNA
1. Messenger RNA (mRNA) - copies DNA's information and takes it out of the
nucleus to a ribosome
2. Transfer RNA (tRNA) - picks up and transfers Amino Acids to the ribosome
* contain an amino acid at one end and an anti-codon at the other
3. Ribosomal RNA (rRNA) - one of the components of ribosomes helps to build the
polypeptide chain/protein
RNA Structure & Function
Protein Synthesis
DNA RNA  PROTEIN
* Two main stages of Protein Synthesis
1. Transcription
* Occurs in the nucleus
* DNA  mRNA
2. Translation
* Occurs at a ribosome
* mRNA  Protein
Transcription
TRANSCRIPTION- Copying a gene from DNA to RNA
Steps
*Binding/Initiation
1. Helicase splits DNA molecule
2. SSBP's hold DNA open
3. RNA Polymerase attaches to ONE of the DNA strands at a promoter region
a. Promoter region - TATA box
*Elongation
4. RNA polymerase builds a complementary mRNA strand in a 5' to 3' direction
a. Reminder: If there is an A in the DNA, a Uracil will be paired to it (no
Thymine in RNA)
*Termination
5. RNA stops when it reaches a termination sequence
a. sequence usually involves 'AAAAAAAA'
6. mRNA transcript detaches and DNA strands go back together
Transcription
mRNA Processing
mRNA must be processed before leaving the nucleus because it contains ‘junk’
* Steps
1. mRNA transcript receives a cap and tail
a) 5' cap - cap of repeated G's at 5' end
b) 3' poly- A tail - tail of repeating A's at 3' end
* These protect the molecule and control its transport out of the nucleus.
2. Introns are excised and Exons are stitched together
a) Introns - pieces of mRNA that are not needed are excised by
spliceosomes and stay IN the nucleus
b) Exons - needed pieces of mRNA that code for proteins. They EXIT the
nucleus and are Expressed. Exons are glued together by SnRP's
(small nuclear ribonucleoproteins)
* The finished product leaves the nucleus and joins w a ribosome to make proteins. The
processing allows several proteins to be made from the same mRNA.
mRNA Processing
Transcription and
Translation cartoon video
https://www.youtube.com
/watch?v=rKxZrChP0P4
Transcription real time video
https://www.youtube.com/watch?
v=SMtWvDbfHLo&list=PLRSu0DZ2i
6tGdGB8reN_yOyfkGq_AX2mE&ind
ex=3
Genetic Code
* Central dogma- all organisms on earth have nucleic acid (DNA/RNA)
that codes for proteins.
* Every three “letters” of mRNA is called a codon. Each codon codes
for one specific amino acid in the protein that will eventually be made.
DNA:
TAC TGC CTC GAA GCC TCG ATC
mRNA:
AUG ACG GAG CUU CGG AGC UAG
Protein:
AUG  Methionine (always at start of chain)
ACG  Threonine
GAG  Glutamic acid
CUU 
CGG 
AGC 
UAG 
Translation
* Steps of Translation - (now out of the nucleus)
1. Binding/Initiation
a. mRNA & tRNA bind to small ribosomal subunit (40sv)
b. Smaller ribosomal subunit binds to larger one (60sv)
2. Elongation
a. tRNA molecules bind to mRNA based on matching their anti-codon to
mRNA codon
b. Amino acids are dropped off and hooked together via peptide bonds
c. Ribosome moves along mRNA, repeating process of bringing tRNA's in,
forming peptide bond, and releasing tRNA's
3. Termination
a. Ribosome reaches a stop codon and the ribosomal complex breaks
apart
* Ribsome has
three sites
1. A site: where
the tRNA is
accepted/attaches
2. P site: where
the polypeptide
(protein) is built by
making a peptide
bond
3. E Site: where
the tRNA exits the
ribosome
Translation
* A gene must start with a start codon (initiator) that
tells ribosome to start translating (AUG - methionine)
* A gene will end with a stop codon (terminator) that
tells the ribosome to stop translating
* There are 3 stop codons (no amino acids) (UAA,
UAG, and UGA.
* Many ribosomes can translate the same mRNA
transcript at the same time
Transcription and
Translation cartoon video
https://www.youtube.com
/watch?v=rKxZrChP0P4
Translation real time video
https://www.youtube.com/watch?v=TfYf_rPWUdY
&list=PLRSu0DZ2i6tGdGB8reN_yOyfkGq_AX2mE&i
ndex=4
Translation
Products of Translation
* Proteins usually
have to be processed
before they can be
used as well. This
occurs at the ER
* Proteins made on
free ribosomes will
be used within the
cell
* Proteins made on
ribosomes are sent to
Golgi to be shipped
out of the cell or used
in the cell membrane
Protein
Synthesis
Overview
Gene Mutations
Mutation - a change in DNA or chromosome
* Causes (mutagens)
* Environmental factors
* Chemicals, sunlight
* Replication errors
* Point mutation- impacts only one base (this is the most common type of mutation)
* Substitution- a base is replaced with another
* Insertion- a new base is inserted – shifts the reading frame
* Deletion- a base is removed – shifts the reading frame
* Non-sense mutation - inserts a stop codon that prevents a protein from being made
* Mis-sense mutation - changes the protein being made
* Silent mutation – Change in DNA, but no change in protein due to ambiguity of code.
Mutations
* Gene Mutations are usually neutral or have no affect.
There are two reasons for this:
1. Mutations that occur in introns will not be expressed in the protein being made.
2. Mutations that affect the third letter in a codon often don’t change the amino
acid
(Wobble effect)
* Mutations can be harmful or beneficial
* EX: make a protein work more efficiently.
* EX: make a protein inactive.
* Mutations are an important source of new genetic variety.
This is where new traits come from.
DNA Mutations and Protein Synthesis
https://www.youtube.com/watch?v=GieZ3pk9YVo&index=2&lis
t=PLIIJseG3DYUK0ijyvRoNqjMia9OvPNVjJ
Gene Mutations
* Recessive disorders
* Sickle Cell Anemia
* point substitution of A for T on Beta-globin gene
* Cystic Fibrosis
* mutation in the gene cystic fibrosis transmembrane conductance
regulator (CFTR) gene
* most common mutation is a deletion of three nucleotides that
results
in a loss of the amino acid phenylalanine
* Hemophelia - MANY causes
* Tay-Sach's
* mutations of the HEXA gene on chrom. 15
* By 2000, more than 100 different kinds of mutations had been
identified in the HEXA gene
* Phenylketoneuria
* mutation of PAH gene on chrom. 12
* More than 400 disease-causing mutations have been found in PAH gene.
Gene Mutations
* Dominant disorders
* Huntington's
* All humans have the Huntingtin gene (HTT) on chrom. 4, which codes for the
protein Huntingtin (Htt).
* Part of this gene is a repeated section (CAG) called a trinucleotide repeat
* it varies in length b/w individuals and may change length between generations.
* When the length of this repeated section reaches a certain point, it produces an
altered form of the protein, called mutant Huntingtin protein (mHtt)
* This leads to increase in decay rate of certain neurons
* Leads to disease symptoms
* Affects muscle coordination and leads to cognitive decline and dementia
Gene Regulation
Every cell in your body contains the same DNA, but some genes are shut off.
~For example, your eyeball cells don’t need the same proteins as your
liver cells.
~In humans these genes can be shut off by methylating them. There are
also regulatory genes that can promote or inhibit the transcription
of certain genes.