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