Exam II (lecture 9-13) Flashcards
Eukaryotic nuclear chromosomes
Chromosoomes are linear and usually very long
- many origins per chromosomes
- slower replication
- shorter okazaki fragments
Histones
Centrpmeres
Linear chromosomes have “end replication problem”
DNA replication takes place during what phase?
S phase, which is part of the interphase of the cell cycle.
Two identical chromosomes are produced, attached at the centromer
Yeast origin
ARS (autonomous replicating sequence)
Creating replication forks at ARS
- origin selection - formation of prereplicative complex (pre-RC) during G1 of cell cycle
- Origin activation - at the beginning of S phase
Step 1 origin selection:
- origin (or replicator) - ARS (in yeast) (autonomously replicating sequence
- Eukaryotic initator - heteohexamer ORC (origin recognition complex)
- ORC - (trans acting factors) recognize and bind to ARSs (cis acting elements). ATP is required for ORC binding.
ORC vs ARS
ORC - trans acting factors
ARS - cis acting elements
Formation of a prereplication complex (pre-RC) in G1.
- binding of initator (ORC)
- Binding of helicase loaders (Cdc6 and Cdt1)
- “Loading of helicases” (Mcm2-7)
Pre-RC consists of
ORC, Cdc6, Cdt1, Mcm2-7
Formation and activation of preRC is controlled by
Cdk (cyclin dependent kinase)
PreRC (origin) activation
S phase
- Cdk and Ddk (cyclin dependent kinases - inactive in G1) activate PreRCs
- Phosphorylation of several proteins leads ro DNA melting and protein recruitment.
- Phosphorylation by S-phase cyclin kinases is necessary for replication fork assembly and confines the initiation of replication to S phase.
Cdk activity low
G1 phase
Pre RC formation allowed
Cdk activity high
S phase
Existing PreRC activation
CDK phosphorylates helicase loaders, preventing them to bind to ORC
When are histones synthesized
only during S phase and are added as replication proceeds
Some histone parts are “inherited” some are new
The spacing of histones every 200nt might be the reason for shorter okazaki fragments in eukaryotes and the slower speed of replication
The “end replication problem” (in eukaryotes)
both ends will be affected!
Results in incompletely replicated DNA due to okazaki fragments.
When completing DNA replication, the lagging strand will have okazki fragments, and the RNA primer fragments have to be removed from the strand by polymerase alpha. This leads to a shorter strand in the end of the newly synthesized DNA.
https://www.youtube.com/watch?v=wf6QiIlGxSg
Telomeres are the ends of the chromosomes
- Protect ends
- Maintain length
Telomere sequence identified as
Simple tandem repeats: TTGGGG
Tetrahymena Thermophila: Single celled pond animal with 40,000 short, linear chromosomes
Telomere repeats in humans
TTAGG
Telomere shortening leads to
cell death or cell senescence
Telomerase reverse transcriptase (TERT)
carry a tighlty bound, noncoding telomerase RNA (TR)
Telomerase:
TERT-TR holoenzyme (10ptn subunits + RNA of 451 nucleotides)
what type of protein is telomerase
ribonucleoprotein
- RNA component from 150-1300 nt long. Serves as a template (3’AAUCCCAAU…5’)
- Protein component has catalytic activities
1. DNA polymerase - adds dNTPs to 3’ end of a linear DNA
2. Reverse transcriptase, that carries its own RNA template
3. DNA/RNA helicase activity
DNA polymerase adds
dNTPs to 3’end of a linear DNA
Reverse transcriptase
carries its own RNA template
DNA/RNA
helicase activity
Telomerase solve the “end replication problem” in eukaryotes
- telomerase extends the 3’ end of the TEMPLATE strand, by adding dNTPs
- Telomerase is a DNA pol and a reverse transcriptase
- Telomerse is very different from other DNA pols: own template, and it synthesizes ssDNA
- After adding a nt repeat, telomerase seperates the RNA-DNA hybrid and repositions on the telomere for extension of the next repeat
- The telomerase-extended 3’ ssDNA terminus is converted to duplex DNA by the same priming and polymerization machinery used in chromosomes replication.
Telomerase characteristics
- is a DNA polymerase and a reverse transcriptase
- is very different from other DNA polymerases: own template and it synthesizes ssDNA
Telomeres replinished by
telomerase
Active: stem cells, germ cells
detectable: many normal adult cell types (quantifiable activity)
In humans ^
Telomeres shorten
during ageing of human fibroblasts
Long telomeres (before telomere shortening)
- Maintenance of genome stability
- Robust proliferation capacity
Telomerase activity
Low oxidative stress
Antioxidant vitamin intake
Psyvhological wellbeing
Physical Activity
Short telomeres (after telomere shortenig)
- Loss of productive structures
- DNA damage responses
- Limited replication potential
- Cellular senescence
Ageing
Stress
Obesity
Smoking
Viral infections
Chronic inflammation
Hormone imbalance
Acohol consumption
Air pollution
Abnormally short telomeres (associated with disease)
Premature aging syndromes
- diabetes
- osteoporosis
- Impaired function of the immune system
- cardivascular disease
Clue to longetivity of telomeres
“Fountain of youth”?
Telomeres are caplike features at the ends of chromosomes that help protect them when cells divide
Over time, due to ongoing cell division, telomeres become shorter. Telomere length appears to be an indication of age and the general health of an individual
Activation of telomerase
Increased incidence of tumors
- maintenance of telomeres are essential to all cell growth
- Inhibiting telomerase would theoretically kill cancer cells (they are immortal)
Telomeres are protected and regulated by proteins: Telomere binding proteins (in mammals, Shelterin)
Sheltrin
- regulate telomerase activity (prevent telomeres from growing abnormally long) and protect chromosomes from joining and exonucleases
Telomere Loop (t-loop)
(protection of telomeres)
- T-loop buries the 3’ terminus of the telomere which further regulates telomere length.
- 3’ end not accessible
Telomeres are requited for
chromosomes protection
Telomerase is essential for
telomere maintenance
telomere shortening leads to
cell death after many cell divisions
short telomeres limit
the growth of cancer cells
short telomeres limit tissue renewal and
contribute to age-related degenerative disease
Mutations are …
changes in DNA sequence that is propegated through cellular generations
When mutations occur in germ line cells
the changes are inheritable
Some frequency of mutation
is necessary to produce the variability on which natural selection acts to drive evolution
Mutations can occur through many different mechanisms, but
all originate as an alternation in DNA
Only after the alteration is converted through replication into an incorrect base pair…
such as AT where, GC should be, does it become a stable inheritable mutation.
The vast majority of damaged nucleotides that occur in a mammalian cell every day..
are repaired by DNA repair enzymes
Point mutations
single base pair substitutions
Mutations of one or few base pairs usually result from
errors in replication or damaged nucleotides
transitions are nearly
10 times more frequent than transversions
Transition examples
Purine to purine (A to G, or T to C)
transversion examples
purine to pyrimidine (A and G turn into C and T)
Pyrimidine to purine (C and T turn into A and G)
point mutations have an effect on
the protein sequence
- A point mutation in the protein coding region of a gene can result in an altered protein with partial or complete loss of function
- Point mutations are known to cause a wide variety of human diseases
- Point mutations in a protein-coding region can be classified by their effect on the protein sequence: Silent, missense and nonsense mutations
Silent mutation
TTT - AAA
lysine stays lysine
Nonsense mutaitons
creation of a stop codon.
Missense mutaiton
formation of a new amino acid.
DNA top strand
= coding = sense = non-template
DNA Bottom Strands
= template strand = antisense = noncoding.
RNA has same sequence as DNA
top strand; is complementary to DNA bottom strand
The genetic code
The DNA sequence encoding a protein is read in triplets or codons
61 of 64 codons are
sense codons for amino acids
3 of 64 codons are
nonsense or termination / stop codons.
AUG codon significance
served as a start codon
the genetic code is degenerate
a single AA can be encoded by more than one codon (20 AA)
Stop codons examples
UGA
UAG
UAA
Nonoverlapping
triplets are ready from a fixed point
open reading frame (ORF)
a run of sense codons (more than 25) before a stop codon is encountered
Silent mutations
- mutations in non-coding and or/non-regulatory regions
- Mutations in coding region - because of code degeneracy may result in the incorporation of the same amino acid
Ecample: A change of TTC (mRNA: AAG) to TTT (mRNA: AAA) is silent because both are codons for Lysine (they are synonymous)
Missense mutation
any mutation within coding region leading to a change of one amino acid for another
- Severity will depend on the nature and the “location” of the change.
sickle cell anemia caused by
missense mutation in the gene that encodes for the beta subunit of the protein hemoglobin
Beta 6th position (Glutamic acid - Valine)
Normal hemoglobin
Function: molecules do not associate with one another; each carries oxygen
Sickle cell hemoglobin
Function: molecules interact with one another to crystallize into a fiber; capactiy to carry oxygen is greatly reduced.
Nonsense muations
- Changes in nucleotides, leading to stop codon.
- Stop codon terminates translation and generates a truncated protein, without a complete AA sequence.
- The severity of a nonsense mutation will depend on the location
Point mutations : a 2 step process
- DNA polymerase incorporates an incorrect nucleotide
- If the mismatch is not required, it becomes a mutation in a second step during replication
Insertion and deletions are
indels
Insertion
occurs when one or more base pairs are added to the wild type sequence
- may have no effect if not in genes or regulatory sequences
- leads to usually a truncated protein
deletion
due to loss of one or more base pairs
- may have no effect if not in genes or regulatory sequences
- leads to usually a truncated protein
indel mutation
of only one or two base pairs in the coding dequence of a protein throws off the reading frame after the mutation, resulting in frameshift mutation.
Indels of 3 or multiple of 3 nucleotides
- preserve the reading frame
- most common: insertion of 3 nucleotides (template slippage by DNA pol during replication)
indels are caused by
aberrant recombination or by templare slippage by the DNA polymerse during replication
Insertion of triplet sequences and human disease
- Triplet expansion diseases
- More than half of the triplet expansion diseases: expansion of the CAG codon for glutamine (Q) - polygrutamine (PolyQ) diseases
DNA alterations that leads to mutations
- Spontanous hydrolysis (water)
- Oxidative damage (reactive oxygen species - ROS)
- Alkylation (alkylating agents)
- Radiation (UV light, X rays , etc)
Spontaneous DNA damage by hydrolysis
Deamination (C,G or A) or loss of bases by hydrolysis
Deamination
removal of an amino group - changes “identity” of bases and their pairing properties
Cytosine to uracil
5-Methylcytosine to thymine
Approx 5% Cs in higher eukaryotes are 5-mC
Deamination of G or A
much less frequent (approx 100x) and produces “abnormal bases”
Guanine becomes Xanthine
Adenine becomes Hypoxanthine
Loss of bases by hydrolysis
depurination much more frequent than depyrimidaiton
1 in 10^5 purines (per 10,000 mammalian cell) are lost from DNA every 24 hrs.
Oxidative damage and alkylating agents can create
point mutations and strand breaks
Deamination by nitrous acid
Sodium nitrate (a common food preservative) as well as nitrosamines are converted to nitrous acid in the stomach.. nitrous acid is a potent mutagen.
Oxidative DNA damage
- possibly the most important source of mutagenic alterations in DNA
- ROS: hydrogen peroxide (H2O2), hydroxyl radicals (OH-), superoxide radicals (O2-), arise during irradation or as byproducts of aerobic metabolism
- Oxidative damage includes modification of bases, sugar, removal of bases and strand breaks.
8-oxoG
Guanine -> 8-Oxoguanine
8oxoG is very mutagenic because it can pair with A leading to _ if not repaired prior to replication
Transversion
Most common mutation found in human cancers
G:C to T:A transversion
Damage by alkylation
addition of an alkyl group, usually to bases
Alkyl groups
- methyl
- ethyl
- propyl
- isopropyl
O^6-MethylG is very mutagenic becuase it tends to pair with T instead of C leading to _ ?
transition
Benzo[a] pyrene (intercalating agent, carcinogen)
smoke of brunign cigarettes, wood, and coal.
In the liver, it becomes a reactive epoxide that can react with bases.
Nitrogen mustard gas (damage by alkylation)
Cross linking agents that react with adjacent G residues preventing replication and transcription
DNA damaging agents are used in Cancer chemotherapy
DNA reactive agents used in chemotherapy for cancer kill cells (cytotoxic) by creating broken chromosomes or stalled replication forks, either of which leads to cell death during cell division.
Ames test
What does it test for?
This test is used to determine mutagenic potential of chemicals in animals
Identifies DNA damaging chemicals
The Ames test for mutagens and carcinogens
- Use histidine auxotrophic (his) Salmonella typhimurium - mutation in biosynthetic pathway for histidine (requires histidine in the growth medium)
- look for His+ revertants
reversion mutation
can synthesize his and grow in his-free medium)
Chemical is a mutagen!
Most known human carcinogens result in
increased mutation in the Ames Test
compounds identified as mutagens in an ames test require…
further testing to determine whether they are likely to be human carcinogens.
Ionizing radiation
UV light (in sunlight), cosmic rays, X-rays
UV and other ionizing radiation
about 10% of all DNA damage caused by environmental agents
UV can cause photochemical fusion of adjacent pyrimidines (pyrimidine dimers - covalent cross links)
Xrays and Gamma rays (higher energy)
Single or doubled strand DNA breaks
Skin cancer major risk
A major risk is prolonged exposure to ultraviolet (UV) radiation.
- Just one indoor tanning session can increase your melanoma risk
- ## Every time you burn or tan, you increase damage of DNA. the more you dmage your DNA, the greater your risk of getting skin cancer.
Deadliest form of skin cancer
Melanoma
Tanning beds
- 97% of women diagnosed with mleanoma before the age 30 have engaged in indoor tanning
- Just one indoor tanning session before the age of 35 increases a person’s risk of melanoma by 75%
- Tanning - indoors or with the sun, makes your skin age more quickly
Squamous cell carcinoma (SCC)
- one tanning session causes a 67% increased risk of developing SSC
Basal cell carcinoma (BCC)
one tanning session causes a 29% increased risk of developing BCC
Summary: DNA mutations
- Hydrolysis can deaminate nucleotide bases, altering their ability to base pair and leading to mismatch during replication
- hydrolysis can also sever the glycosyl bond between the pentose and the base, leaving an abasic site
- Nitrous acid, the metabolic product of food preservative, can induce the deamination of A or C residues, resulting in a transition mutation
- Oxidative damage is caused by reactive oxygen species that react with nucleotides at many different positions in the molecule. Oxidation can affect base pairing or cause DNA strand breaks
- Alkylating agents attack DNA and add bulky chemical groups to the base or the ohosphodiester backbone. Alkylation can alter the base pairing of the nucleotide
- DNA damaging agents can lead to muations at low concentrations and can kill the cell ay high concentrations
The ames test determines whether a compound is
mutagenic in bacteria, thus identifying the compound as a potential carcinogen
UV light from the sun
can form pyrimidine dimers, that stall DNA polymerase during replication
Mismatch repair (MMR)
involves a removal of several to many nt’s from new strand
- Photoreactivation
photorepair of cyclobutane pyrimidine dimers
DNA photolyase
uses the energy derived from absorbed visible light to reverse damage of UV light
Photolyases: present in all almost all cells
Bacterial, archael and eukaryotic. Yet, for some reason are not present in the cells of placental mammals (including humans).
chromophore
light absorbing group
Direct reversal of DNA damage (Direct repair)
- Photoreactivation
- Removal of alkyl groups
removal of alkyl groups
repair oxidized nucleotides (O^6-methylguanine). Requires specific enzymes
O^6 methylguanine-DNA methyltransferase
catalyzes transfer of methyl grpup of O^6 methyl guanine to one of its own Cys residues
O^6 methylG is very mutagenic because it tends to pair with T instead of C, leading to_?
Base Excision Repair (BER)
Functions at the level of single damaged nucleotide and it is involved in several types of repair mechanisms.
- Removal of single “damaged” base (alkylation, oxidation, or deamination); most U’s are removed by BER
- Removal of abasic sugar (recognition and repair of AP sites)
- This is a main mechanism for repairing of single strand breaks that don’t have the proper ends for ligase, “clean up” for ligase
DNA Glycosylase
recognition of the damaged base
Glycosylases scan DNA and “flip”
damaged base out of the helix… and remove it if it fits in the catalytic site
DNA glycolysas - two main types:
- highly specific to a particular damaged base
- Recognizes oxidative damage (diverse substrate spectrum).
Nucleotide Excision Repair (NER)
Targets large, bulky lesions and removes DNA on either side of them.
- Protein complexes recognize a variety of base damages resulting distortions to DNA structure. In contrast to BER, NER does not require specific recognition of a damaged nucleotide.
- NER enzymes cleave damaged DNA strand on both sides of the “lesion” (removing more than the damage itself)
- Specialized helicase “releases” cut fragament
- DNA polymerase fills in the single gap using undamaged strand as a template
- Ligase closes the nick.
- Predominant repair pathway for removing pyrimidine dimers, 6-4 photoproducts, and several bulky base adducts, including benzo[a] pyrene-guanine.
NER in E.Coli
Involves primarily four proteins (UvrA, UvrB, UvrC, UvrD)
- UvrA2 UvB complex scans for NDA distortionsm (damage)
- UvrA leaves
- UvrB melts short stretch of DNA
- UvrC nicks both sides of distortion. (about 12-13 bases) (exinuclease)
- UvrD (helicase) releases the oligonucleotide
- DNA Pol. I fills in and ligase closes the gap.
NER in eukaryotes
Main factors discovered through reasearch on XP patients (Xeroderma pigementosum)
- individuals extremely sensitive to singlight, thousands of times more likelu to develop skincancer
- Most patient develop neurological abnormalities
- Mutations in at least seven genes (XPA-XPG) result in XP (defective NER)
NER is the sole repair
pathway for pyrimidine dimers in humans
TCR
Transcription-coupled repair
Specifically targets repair to actively transcribed DNA
DSBs
(double strand breaks) can be repaired by homologous recombination (error “free”) - typical route
Phases of the cell cycle when no sister chromatids are present –>
NHEJ - (non homologous end joining)
is a repair system for DSBs (double strand breaks) - usually leads to loss of DNA (produce mutations)
UvrA
recognzies lesion
UvrB
Unwinds DNA
UvrC
Exinuclease
UvrD
Helicase
Pol I
fills in gap
DNA ligase
seals DNA
NHEJ
Non homologous end joining
All eukaryotes; detected in some bacteria
Translesion DNA synthesis
lesion at a replication fork after the DNA strands have been unwound
Damaged DNA is encounteres by the fork
Specialized TLS DNA polymerases are employed
TLS DNA Pol:
- error prone: Usually lacks proofreading activty (low fidelity)
- Extends DNA strand across bulky template lesion (often results in a mutation)
TLS polymerases in humans
10
mismatch repair system corrects
nucleotide residues misincorporated during replication
some types of lesions are repaired directly, such as
photoreversal of pyrimidinse dimers by photolyase
The base excision repair pathway corrects relatively small,
single base lesions and uses different DNA glycosylases to recognize particular lesions
nucleotide excision repair system repairs
bulky lesions by using exinuclease that makes strand incisions on either side of the lesion
Transcription coupled repair
adapts the nucleotide excision repair system to lesions identified by a stalled RNA polymerase
Double stranded DNA breaks result in
fragmented chromosomes and are usually repaired by homologous recombination, a highly fidelity process
Double strand breaks can also be processed by
error-prone nonhomologous end joining
Cells contain multiple
specialized DNA polymerases that extend DNA across lesions, but translesion synthesis is usually error-prone process that results in mutation
