DNA replication and Telomeres Flashcards
DNA polymerase
catalyzes the addition of nucleotides to the 3’ end of a growing strand of DNA using a parent DNA strand as a template
also proofreads nucleotides added and removes those that are paired incorrectly
DNA helicase
uses the energy of ATP hydrolysis to unwind and separate the DNA double helix ahead of the replication fork
single-strand DNA binding protein
binds to single-stranded DNA exposed by DNA helicase and prevents base pairs from re-forming before the lagging strand can be replicated (aka keeps DNA double helix from reforming during replication)
DNA topoisomerase
produces transient breaks (aka nicks) in one strand of DNA double helix to relieve the tension built up by the unwinding of DNA ahead of the DNA helicase
reseals break after DNA has relaxed
sliding clamp (protein clamp)
keeps DNA polymerase attached to the template, allows the DNA polymerase to move along without falling off
primase
synthesizes RNA primers so that DNA polymerase can initiate synthesis
DNA ligase
uses the energy of ATP hydrolysis to join Okazaki fragments made on the lagging strand template
what were the three proposed models of DNA replication?
a. Conservative: the original/parental DNA molecule remains intact and an entirely new DNA molecule is synthesized
b. Semiconservative: each of the 2 strands of the parental DNA molecule serves as a template for the synthesis of a new strand
c. Dispersive: the original DNA molecule is fragmented and the daughter molecules consist of a mix of old and new DNA scattered in pieces throughout both strands
Name and describe the experiment that proved that DNA replication is semi-conservative
Meselson-Stahl Experiment
1. labeled DNA with nitrogen isotopes (15N-heavy and 14N-light) and grew DNA in the two mediums
2. used density gradient centrifugation (centrifuged at high speed for 48 hours to form cesium chloride density gradient): 15N-heavy will form a band near the bottom of the centrifuge tube and 14N-light will form a band near the top of the tube
3. after 1 generation, both daughter DNA molecules were of INTERMEDIATE weight
–> demonstrated that DNA replication is semi-conservative
what are replication origins and how do they differ between eukaryotes and prokaryotes?
replication origins: where DNA synthesis begins (typically in TATA regions)
Prokaryotes: only one origin of replication
Eukaryotes: multiple origins of replication; can simultaneously replicate regions of DNA
If DNA polymerase only works in the 5’ to 3’ direction, how do you duplicate both strands simultaneously?
asymmetry of the replication fork (leading - continuous and lagging - fragmented strands)
DNA made on lagging strand is made of short 5’ → 3’ sequences
how far could DNA replication go if the following protein was inactivated and how would the leading strand vs lagging strand be affected?
- DNA polymerase
- DNA ligase
- sliding clamp for DNA polymerase
- nuclease that removes RNA primers
- DNA helicase
- primase
DNA polymerase: RNA primers would be laid down at origin
DNA ligase: Lagging strands would be complete but unconnected fragments
Sliding clamp for DNA polymerase: Slows down the rate of DNA synthesis since DNA polymerase would fall off frequently
Nuclease that removes RNA primers: Lagging strand will have unconnected fragments of RNA and DNA
DNA helicase: Replication could not start since the DNA strands would be stuck together
Primase: RNA primers cannot begin on the leading or lagging strand → no DNA
when, where, and why does DNA replication occur?
when: occurs during interphase (before mitosis or meiosis) before cell divides
where: in nucleus for eukaryotic cells
why: before a cell undergoes division, it needs to get more DNA into the new daughter cells
describe the steps for leading strand replication
- DNA replication starts at the replication origin where helicase comes in to unwind the 2 strands: SSB proteins (single stranded binding) bind to the unzipped DNA strands to make sure they don’t come back together and topoisomerase keeps the DNA strands from getting supercoiled
- Primase makes RNA primers for both strands
- DNA polymerase comes in and begins adding onto the RNA primer, starting replication from 5’ to 3’ direction with no issues (same direction as the unzipping)
describe the steps of lagging strand replication
same as leading strand until DNA polymerase comes in on step 3.
1. primase continues to synthesize new RNA primer and DNA polymerase needs to keep backtracking to build the new DNA strand → new primers need to be placed constantly → creates DNA fragments called okazaki fragments
3. the new okazaki fragments are extended to the previous adjoined fragments
4. nuclease removes previous RNA primers
5. repair polymerase replaces RNA primer with DNA sequences
5. DNA ligase: seals the gaps between the okazaki fragments and glue them together
what is the problem with replicating the lagging strand of linear chromosomes?
lagging strand replication cannot be completed to the end of the chromosome
RNase H removes the RNA primers and then DNA polymerase comes in and adds the appropriate DNA nitrogen bases at the 3’ end → however, the last primer at the end of the lagging strand ends with a 5’ end, so DNA polymerase cannot add DNA there → this results in a shortening of the lagging strand each time replication/cell division occurs since some of the genome is not included in the replication → can lose important genes → this problem is solved by telomerase replication
How is the issue of telomere shortening solved?
the telomerase enzyme has its own primer (AAUCCC) and it elongates the parent strand adding repeating sequences of TTAGGG → the last two bases of DNA strand pair up with the last two bases and then telomerase enzyme drives DNA synthesis to elongate the original/parents DNA strand more (cannot synthesize the newly made strand since it is 5’ end) → the RNA primer lays down another template at the very end of the newly added part of DNA strand → DNA polymerase starts to add DNA to the 3’ end of the primer → back to the same situation where RNase H removes the primer but now FEN1 can remove the overhand flap without losing any actual parent DNA/genome
what is the relation between telomere length and aging?
Telomere length shortening plays a role in aging since there is an innate limit (hayflick limit) to the number of times a cell can divide in its lifetime before it undergoes senescence. This shortening is associated with aging as well as aging-related diseases (there are more senescent cells in older humans than young humans and cells senesce earlier in older humans as well since their telomere lengths are shorter)
telomeres
The protective caps at both ends of each chromosome
How does telomerase contribute to cancer?
Telomerase is the enzyme that extends the telomere sequence at the ends of the chromosomes to prevent the loss of genetic info during replication. It plays a role in cancer since it keeps extending the telomeres and thus chromosome stability increases and the cell never undergoes the normal cycle of senescence, which eventually leads to biological immortality of the cell and unchecked proliferation, which leads to cancer.
What are the major findings of Hahn et al 1999 Nature 400:464?
The first example of transforming normal human cells into tumorigenic cells by adding large amounts of hTERT, a RAS allele, and Large-T. It was the creation of human tumor cells with defined genetic elements. The experiment showed that an increase in hTERT activity leads to an increase in telomere length and thus chromosome stability, which gives way to biological immortalization of Large-T expressing HEK and BJ cells
by co-expressing telomerase (hTERT), simian virus 40 (SV40) large-T oncoprotein (SV40-T Ag), and an oncogenic allele of H-RAS (RAS), normal human cells could become tumorigenic
what is the full name of TERT and its importance of TERT expression and activity in cancer?
Telomerase Reverse Transcriptase
TERT is the catalytic subunit of telomerase. In cancer, TERT expression and activity is increased, which results in cells avoiding the usual senescence and leads to unchecked proliferation and ultimately cancer (most normal somatic cells, except for germ cells and stem cells, do not have TERT expression activated)
how is TERT and telomerase regulated in human cells?
Chromosomes in majority of human somatic cells have a set length of telomeres at their ends → each time cell divides, the telomeres’ length decrease until it runs out → cell undergoes senescence
Majority of somatic cells do NOT have telomerase/TERT activated
most normal somatic cells, except for germ cells and stem cells, do not have TERT expression activated (telomerase is not active in most human cells)
what are stem cells and germ cells?
stem cells: unique cells in the body with the ability to divide and develop (differentiate) into specialized cells with specific functions (can self-renewal for long periods of time –> why they have telomerase activity activated)
germ cells: sperm and egg cells
list and explain an assay (full name) to measure telomerase activity
Telomeric Repeated Amplification Protocol assay (TRAP)
- Cell Extraction: Cell extracts are prepared from the samples (e.g., tissue, cell lines)
- Incubation/telomerase extension: The extracts are incubated with a specific substrate that telomerase can extend (telomerase adds telomeric repeats to a substrate)
- PCR Amplification: The extended products are amplified through polymerase chain reaction (PCR), using primers that bind to the telomeric sequences
- Analysis: The PCR products are then analyzed (usually by gel electrophoresis) to visualize the telomerase activity
list and explain at least one of cellular senescence biomarkers
p16
Clearance/removal of p16 positive senescent cells delays aging-associated disorders; accumulation of p16 + senescent cells is linked to various age-related diseases
High p16 levels: associated with the presence of senescent cells (particularly in aged tissues)
SA-beta-gal
what are the most important components of telomerase?
Telomerase: a ribonucleoprotein complex composed of TERT and TERC: Telomerase Reverse Transcriptase
TERT is the catalytic subunit/component of the enzyme telomerase and is responsible for adding the telomeric repeats to the ends of chromosomes
TERC: Telomerase RNA component
TERC serves as an RNA template that provides the sequence needed for TERT to add telomeric repeats to the chromosome ends
senescence
the process by which cells stop dividing and enter a permanent state of growth arrest without undergoing apoptosis (cell death)
what are the pros and cons of senescence?
PRO: Helps prevent the proliferation of damaged cells (tumor suppression and wound healing)
CON: Accumulation over time contributes to the aging process and the onset of age-related disorders
what is the hayflick limit?
Hayflick limit: the number of times a normal human cell population can divide before it stops dividing and enters a state of senescence (intrinsic limit to the number of cell divisions)
Largely driven by the progressive shortening of telomeres with each cell division
Human cells can divide around 50 times before senescence
telomerase
- RNA dependent DNA polymerase → makes DNA using RNA as a template → reverse transcriptase
- Carries RNA template
- Synthesizes (replicates) telomeric DNA repeats (TTAGGG)
- A ribonucleoprotein complex composed of TERT and TERC
what sequence are telomeres
repeated TTAGGG sequences
okazaki fragments
short sequences of newly synthesized DNA that are produced during the replication of the lagging strand
what direction does DNA synthesis occur in?
5’ to 3’ direction (nucleotides are added on the 3’ end of DNA strand)
what is the primary function of DNA?
provide almost unlimited, accurate copies of the genetic code
what was the NASA twins study?
NASA Twins Study: is a landmark experiment that explored the effects of space travel on human health through a multidimensional approach
study focused on telomere length, chromosomal abnormalities, and other biological changes experienced during spaceflight and compared data from astronaut Scott Kelly (spent nearly a year in space) to his identical twin, Mark Kelly (remained on Earth as a control)
what were the findings of the NASA Twins Study?
Telomere length increased during spaceflight BUT shortened rapidly (returned to pre-flight levels or lower) upon returning to Earth
- CD4 T cells, CD8 T cells, and LD cells shows increased telomere length
- CD19 Cells (B cells) did NOT show increased telomere length
- showed that even after 6 months on Earth, there are MORE SHORT telomeres
Chromosomal inversions and translocations detected in Scott Kelly’s genome after prolonged spaceflight (thought to be related to exposure to space radiation, especially ionizing radiation and other environmental factors)
how many and what types of samples were collected in the NASA Twins Study?
total of 317 biological samples were collected from both twins before, during, and after the space mission
Samples included stool, urine, and whole blood → were further separated into peripheral blood mononuclear cells (PBMCs), sorted immune cells, and plasma for detailed analysis
what were the 10 NASA Twins Study Analyses?
Biochemical
Cognition
Epigenomics
Gene expression
Immune
Metabolomics
Microbiome
Proteomics
Physiology
Telomeres
what are PBMCs and its full name?
Peripheral Blood Mononuclear Cells
definition: Any peripheral blood cells (aka white blood cell) having a single round nucleus
what are PBMCs consisted of?
lymphocytes (T cells, B cells, natural killer NK cells)
monocytes
dendritic cells
what are the subtypes of lymphocytes?
T cells, B cells, and NK cells
what are the subtypes of monocytes?
dendrite cells and macrophages
CD4+ T cells (helper T cells)
coordinate adaptive immunity through activation and regulation of other immune cells
percentage among PBMCs: 25-60%
markers: CD3+ CD4+
B cells
secrete antibodies as part of humoral immune response
percentage among PDMCs: 5-10%
markers: CD19+
natural killer cells (NK cells)
trigger lysis or apoptosis of infected cells without the need for prior activation by a specific antigen
percentage among PDMCs: 10-30%
markers: CD56+ CD3-
monocytes
The largest type of white blood cells in PBMCs and play an
important role in the adaptive immunity process
take up foreign objects via phagocytosis, perform antigen presentation, and produce cytokines
Circulate in the blood and can migrate into tissues where they differentiate into macrophages or dendritic cells
percentage among PDMCs: 5-10%
markers: CD14+
macrophages
macrophages are monocytes that have migrated from the bloodstream
into any tissue in the body
crucial for phagocytosis (ingestion of pathogens and dead cells)
dendritic cells
process and present antigen material to T cells
Act as a bridge between the innate and adaptive immune systems
percentage among PDMCs: 1-2%
what are leukocytes and erythrocytes?
leukocytes = white blood cells
erythrocytes = red blood cells
what are mononuclear white blood cells and their subtypes?
Agranulocytes are characterized by the absence of prominent granules in their cytoplasm and they have a single, large nucleus
T cells
B cells
NK cells
dendritic cells
monocytes
lymphocytes
what are morphonuclear white blood cells (granulocytes) and their subtypes?
granulocytes: have a multi-lobed nucleus and contain granules in their cytoplasm that are involved in the immune response
neutrophils
eosinophils
What does the buffy coat from whole blood contain?
Platelets and leukocytes (WBCs)
specific types of leukocytes include…
a. nuclear WBCs: T cells, B cells, NK cells, dendritic cells, monocytes
b. morphonuclear WBCs: granulocytes (such as neutrophils and eosinophils)
PBMCs are within the buffy coat as well
what are CD markers and what are the specific CD markers for…
T cells
B cells
NK cells
CD (cluster of differentiation) markers are essential surface proteins on various immune cells that help in identifying and characterizing these cells based on their function, development, and activation state
T cells: CD4+, CD8+
B cells: CD19+
NK cells: CD56+CD3-
density gradient centrifugation for cancer tumor cell isolation (CTC)
The most widely used approach for the isolation of CTC is density
gradient centrifugation in the presence of different commercially available reagents (ie. Ficoll.
This method is based on the lower density of CTCs and nucleated
blood cells and uses a density gradient medium to separate these cells of similar density from other blood constituents
how is PBMC isolated from whole blood?
PBMC isolation from whole blood using density gradient
separation (using density media, Ficoll or Histopaque)
What do the layers look like in PBMC isolation via density gradient separation?
top to bottom of tube (lighter things at the top)
1. plasma: platelets, cytokines, hormones, electrolytes
2. buffy coat: PBMCs, leukocytes (WBCs), monocytes, progenitor cells
3. morphonuclear cells (eosinophil and neutrophil) and erythrocytes (RBCs)
What do the layers look like in Cancer Tumor Cell (CTC) isolation via density gradient separation?
top to bottom
1. tumor cells
2. platelets
3. leukocytes (WBCs)
4. erythrocytes (RBCs)
CD8+ T cells (Cytotoxic T cells)
responsible for killing cancer cells and cells that are virus-infected or damaged
percentage among PDMCs: 5-30%
markers: CD3+ CD8+
how can you categorize white blood cells via nucleus?
mononuclear WBCs (PBCMs: T cells, B cells, NK cells, dendritic cells, monocytes)
morphonuclear WBCs (aka granulocytes): neutrophils and eosinophils
difference between PBMC isolation and CTC isolation?
PBMC isolation: immune cells
CTC isolation: rare circulating cancer cells
What is the commercially available reagent commonly used for density gradient centrifugation?
Ficoll
what is the function of DNA?
to provide almost unlimited, accurate copies of the genetic code
double strand DNA (dsDNA) has what property?
the two strand run ANTIPARALLEL to each other (5’ to 3’; 3’ to 5’)
Questions: What are the distributions of 14N and 15N in the DNA after successive
rounds of replications?
* 3 rd generation? _______HL , ______LL
* 4 th generation? _______HL , ______LL
3rd generation: 1/4 HL, 3/4 LL
4th generation: 1/8 HL, 7/8 LL
What are some properties/facts about replication origins in eukaryotes?
- replication origins typically in TATA regions
- humans have 10,000 origins
- DNA replication can happen in BOTH directions (replication fork) left and right
- can replication 2 DNA strands SIMULTANEOUSLY (leading and lagging)
what are the rates of nucleotides added per second for humans vs bacteria?
humans add 100 nucleotides per second
bacteria add 1000 nucleotides per second
where does DNA polymerase get the energy to catalyze the addition of a nucleotide to the growing DNA chain?
uses the energy from the tri-phosphate bonds on the incoming nucleotide
what is the repeated sequence of telomers?
TTAGGG (on the other strand, CCCTAA)
Why did Carol Greider win a Nobel Prize in 2009?
She proved that telomere lengthening activity is due to a unique telomere-synthesizing enzyme (telomerase)
- telomerase is required for cells that undergo many rounds of divisions, especially tumor cells and some stem cells
what is whole blood composed of?
erythrocytes (red blood cells), leukocytes (white blood cells), platelets
What is a distinction between the buffy coat and PBMCs?
buffy coat contains both the mononuclear and granulocytes (contains cells with both 1 nucleus and multi-nucleus); but PBMC only has the mononuclear cell fraction
clamp loader
uses the energy of ATP hydrolysis to lock the sliding clamp onto DNA
