Exam 3 Flashcards
What is the shape of bacterial chromosomes?
Bacterial chromosomes are circular (often bacteria have both genomic DNA and plasmids; circular-replicated and segregated at cell division)
What are Chromosomes and why is size so important?
- Very large; genomic DNA/RNA longer than what they are found in so must be compacted
- Chromosomes themselves are macromolecular entities that must be synthesized, packaged, protected, and properly distributed amongst daughter cells at cell divisions
what is the shape of virus chromosomes?
Viruses (genomes made of DNA or RNA; circular or linear) (single or double-stranded)
What is the most common shape of eukaryotes chromosomes?
linear (vary in number depending on species)
Which organelles contain their own circular genome?
Mitochondria and chloroplasts
What types of cells are most common in humans and when is it not?
most cells are diploid except when they are haploid seen in gametes
Describe chromosome lengths in nature.
-Longer than the cellular or even viral packages containing them
-Eukaryotes: A yeast cell, one of the simplest eukaryotes, has 2.6 times more DNA in its
genome than an E. coli cell. Cells
of Drosophila melanogaster, the fruit fly used in classical genetics studies contains more than 35 times as much DNA as E. coli cells, and human cells have almost 700 times as much.
-Typical cell is 7-30 um across; nucleus 10 um diameter
-Diploid Human DNA stretched 2m from one cell (2million um)
What are the 3 DNA sequences dedicated to the maintenance of chromosomes?
*Replication: initiation and termination
*Origins of replications (ORIs)
*Segregation during cell division (centromeres which are important for mitotic events)
*Protection of chromosomes: telomeres (specialized DNA sequences at the ends of chromosomes)
What is a centromere?
A segment of each eukaryotic chromosome that is AT-rich and functions during cell division as an attachment point for proteins that link the chromosome to the mitotic spindle at metaphase
Why is the centromere important to the chromosome?
-This is necessary for the orderly and even distribution of chromosomes in daughter cells
-Biding sites for cen proteins( centromere proteins)
-ESSENTIAL FOR STABLE SEGREGATION OF CHROMOSOMES DURING CELL DIVISION
What is a telomere?
Telomeres are sequences at the ends of eukaryotic chromosomes that add stability by protecting the ends from nucleases and providing unique mechanisms for the faithful replication of DNA
Why is telomere important for chromosomes?
- This is necessary because DNA polymerases have a built-in problem during synthesis where base pairs get cut out
- Telomeres bind to specialized proteins needed for proper function
long sets of nucleotides that work like repeating sequences surrounded by proteins
* 5’-(TxGy)n ;x and y are generally between 1 and 4
* N is 20-100 in most single-celled eukaryotes but 1500 in mammals
Explain the idea of telomere repeating sequences
long sets of nucleotides that work like repeating sequences for example (TTAGGG)xn is the fragment that continues to repeat at the ends of chromosomes where proteins create protection
* 5’-(TxGy)n ;x and y are generally between 1 and 4
* N is 20-100 in most single-celled eukaryotes but 1500 in mammals
Which are smaller Mitochodniral DNA molecules or nuclear chromosomes?
Mitochondrial DNA molecules
what is a larger animal or plant mt DNA
plant
how does chloroplast DNA exist
cpDNA circular duplexes 120 to 160 kbp
organelle DNA undergoes ?
considerable compaction like nuclear DNA
what does mtDNA encode for in the mitochondrial
tRNAs and rRNAs and a few proteins that are necessary for cellular respiration.
what are 95% of mtProteins encoded by ?
nuclear DNA
about 37 genes of 20,000 are encoded by?
mitochondria
What is also passed on when cell division occurs?
Mitochondria and chloroplasts divide when the cell divides. Their DNA is replicated before and during
cell division and the daughter DNA molecules pass into the daughter organelles.
How is mitochondria DNA inherited?
maternally in humans and other organism
what are some theories about why mitochondrial DNA is maternal?
- We don’t fully understand why—lots of theories
- Sperm mitochondria self-destruct upon
fertilization? (shown in C. elegans) - Autophagosomes, for instance, are known to engulf paternal mitochondria shortly after a sperm penetrates an egg.
- Some evidence that there is paternal mitochondrial DNA in some individuals?
what genetic testing has been spawned by maternal inheritance?
services like 23andMe to trace our maternal ancestries.
DNA replication is very complex. true or false?
true
DNA replication is similar between which domains?
bacteria archaea and eukaryotes it’s highly conserved
what two steps are key for the regulation of DNA replication?
Initiation and termination
What type of mechanism is replication and its direction?
semiconservative directionality of 5’-3’ bidirectional from origins
where does replication begin?
ORI ( origin of replication)
Enzymes and chemistry make up?
DNA polymerase
DNA replication mechanism has?
replication fork and lagging versus leading strand synthesis
What are the 3 proposed models for replication?
Conservative, semiconservative, Dispersive
Explain how it was discovered semiconservative was the right system for DNA replication.
- By using isotopes N14 ( labels light DNA) and N15 which labels heavy strands of DNA you can place them in a CsCl solution to cause separation by mass.
- In the first generation by replicating DNA and centrifuging it the observed result was a single layer separation due to equal portions of Parent and first replication existing together lead semiconservative or dispersive being possible explanations
- In a second-generation experiment the observed effect is the formation of two clear layers of light and mix which corresponds to the semiconservative idea for DNA replication.
Conservative model
DNA replication leads to the original strands remaining together
-In 1st generation, we see 2 DNA duplexes 2 old together and 2 new together
-2nd generation 4 duplex form where we have 2 old strands together and the formation of a duplex that is all new
semiconservative model
There is a mixture of strands between old and new
-1st generation old and new for two new duplex
-2nd generation the formation of two duplexes between old and new strands and the formation of two new duplexes made up of new strands
Could density gradient determine or distinguish where synthesis was initiated on a chromosome?
No, they could not
How was ORI detected?
using replication in bacteria ( plasmid with radioactive e DNA and electron microscopy
Does DNA completely unwind during replication?
NO
Replication fork
The point where the parental duplex separates and the daughter duplexes form was the site of new DNA synthesis
bidirectional replication
two replication forks are formed at the origin and propagate away from it in opposite directions.
why is plasmid a good model?
The same replication process in the bacterial chromosome as in the plasmid
What does the replication fork consist of?
The Y-shaped replication fork consists of a parental duplex DNA stem and two prongs, the new daughter duplexes.
What is the direction of the DNA strands in the fork?
The parental DNA strands are antiparallel, so the links between nucleotides in the two daughter strands also run in opposite directions.
What direction does DNA polymerase enzyme synthesize?
extend DNA in only one direction 5’-3’
Why does the limited directionality of DNA polymerase cause a hiccup in replication?
*both daughter strands cannot be replicated in the same direction in which the replication fork moves.
* DNA polymerase cannot initiate DNA chains; these must be initiated by primers. In the cell, the primers at a replication fork are RNA, synthesized by an enzyme called primase.
What is the direction of synthesis DNA polymerase a reference to?
the direction in which each new nucleotide is chemically linked to the growing daughter strand.
how does the DNA polymerase function of linking happen?
*by linking the α-5′- phosphate of a new dNTP to the 3′ position of the nucleotide residue at the end (i.e., the 3′ end) of the chain.
*Thus, the directionality is 5’-3’ in terms of the growth of the strand
Semidiscontinous
- Only one daughter strand is synthesized continuously; the other is made as a series of discontinuous fragments.
* The strand that is made in the direction opposite to fork movement is still synthesized in the 5′→3′ direction, and thus the strand must be reinitiated at intervals and synthesized as a series of fragments
leading strand
the continuously synthesized daughter stand
lagging strand
the discontinuous daughter strand
what is the idea of lagging based on?
The “lagging” designation is based on the fact that some unreplicated single-stranded DNA is generated on the lagging strand by the moving fork, so the conversion to duplex DNA on this strand lags behind that of the leading strand.
How were Okazaki fragments found?
- Okazaki used E. coli cells infected with T4 phage.
- Because T4 makes many copies of itself simultaneously, detection of lagging-strand fragments is made easier by their abundance.
- T4-infected E. coli cells were labeled with radioactive nucleotide precursors for brief time intervals, then the DNA was analyzed in an alkaline CsCl gradient to separate the new radioactive DNA strand from the unlabeled parental strands
- Small fragments in the 1,000 to 2,000 nucleotide (1 to 2 kb) range were observed, as predicted, and have come to be known as Okazaki fragments.
What is the size of Okazaki Fragments?
1-2kb long in bacteria but are shorter about 100 to 200 nucleotides in eukaryotes
how is the Okazaki fragment primed?
at the 5’ end by a short RNA of 10-13 nucleotides long.
RNA primer is removed by?
nuclease action and the Okazaki fragments are joined by ligase soon after the replication fork has passed.
When was the first DNA polymerase discovered?
1950s
What was the original discovered DNA polymerase?
E.coli DNA polymerase I
how many polymerases are in E.coli?
5
Pol 1 mainly functions as a?
damage repair and connecting Okazaki fragments
describe the direction of the exonuclease of each 3’-5’ and 5’3’
Pol 1
Pol 2
Pol 3
Pol 4
Pol 5
- both
- 3’-5’
- 3’-5’
- none
- none
functions of the 5 Pol
pol 1 - Ozaki fragment processing (remove RNA primers and fill gaps with DNA)
pol 3- chromosome replication
pol 2,4,5- translesion synthesis (DNA repair)
what guides the polymerization reaction in DNA polymerase?
template strand
why is the primer key for polymerase to work?
The primer is required because polymerase adds dNTPs to the free hydroxyl group at the 3’ OH at the end
primer is RNA
Explain the reaction occurring during the addition of a dNTP
- All nucleic acids grew by addition at 3’-end, not at 5’-end;
- 3’ OH reacts with Phosphate of incoming NTP; pyrophosphate is released
What does Pol 1 do with incoming dNTPs
Pol I differentiates amongst incoming dNTPs after it undergoes a conformational change that checks for proper geometry of the base pair formed
DNA nucleases
a class of enzymes that degrade DNA
exonucleases
nucleases that shorten DNA from the ends
endonucleases
cut DNA at internal positions
All cells have many nucleases of both types that do various tasks how would a biochemist study the function of a type of enzyme that uses DNA as a substrate?
must carefully purify the enzyme and remove all nucleases
What did Kornberg discover when trying to purify Pol 1 and being unable to remove exonuclease activity?
The same enzyme that makes DNA also degrades it
Describe the 5 steps of correction of improper base pair binding in DNA replication.
- Polymerase mispairs dC with dT.
2.Polymerase repositions the mispaired 3’terminus into the 3’-5’ exonuclease site - Exonuclease hydrolyze the mispaired dC
- The 3’ terminus repositions back to the polymerase site
- Polymerase incorporates the correct nucleotide, dA
Exonuclease that digests a DNA strand from the 3’ terminus is called?
Exonuclease 3’-5’ because the strand shortens at the 3’ end while the 5’ end remains intact
5’-3’ exonuclease
exonuclease digests DNA from the 5’ terminus while the 3’ end remains intact.
In E.coli there is one error in 10^9 polymerization events but replication of 4.6x10^6 bp requires polymerization of 9.2x10^6 dNTPs so one would expect about one error per cell. How many observations of errors are seen?
one error is acquired in every 100 to 1000 new cells
Why are there such small amounts of errors in DNA polymerization?
Error correction is built in but there is also a repair mechanism that comes into play
DNA polymerase structure of E.coli Pol 1 resembles?
right hand
palm, thumb, fingers
All DNA polymerases have the same structural features ( True or False)
True
Explain Pol 1 as the hand structure during polymerase
- The bound DNA lies on the palm domain, which contains the polymerase active site and is the most conserved feature among all DNA polymerases.
- The finger domain contains the dNTP-binding site, and the thumb domain partially curves around the duplex portion of the primed template, tightening the grip on DNA
What causes the polymerase to not close all the way?
Incorrect dNTP
Give an example of Induced fit catalysis in DNA
Accuracy of polymerase functions at the level of shape recognition
Explain how Mg plays a mediation role in DNA synthesis.
- The polymerase active site contains two magnesium ions that are held in place by conserved Asp residues
- One Mg2+ deprotonates the primer 3′-OH group to form the 3′-O− nucleophile.
- The other binds the incoming dNTP and facilitates the departure of the pyrophosphate leaving group.
- This two-metal-ion-catalyzed reaction is remarkable in that no amino acid side chain plays a direct role in catalysis; the two metal ions do it all.
*The 3’-5’ exonuclease uses a similar two-metal-ion mechanism
in 3’-5’ exonuclease, what role does Mg2+ play?
Have the ability to coordinate the removal of the wrong dNTP attachment
What does processivity do in DNA ?
Increase the efficiency of DNA polymerase activity
- When a polymerase remains attached to the DNA during multiple catalytic
-Processivity can result in exceedingly efficient polymerization, because much time is wasted by a
dissociated polymerase in locating and rebinding a 3′ primer strand terminus.
What is a processivity number?
the average number of nucleotides incorporated before the enzyme
dissociates from the DNA
Pol 1 has processivity number of ?
10-100 nucleotides depending nucleotides
Pol 3 has a processivity number of ?
most DNA polymerases that replicate chromosomes has a processivity number in the
thousands, which, as we will see, results from a protein ring that encircles the DNA.
which enzyme does Initiation of replication starts by double helix unwind providing single stranded DNA templates?
Helicases and single strand binding proteins
what enzymes are role players in synthesis of leading strand?
Primase- Priming
DNA polymerase- elongation and Replacement of RNA primer by DNA
what enzymes are role players in synthesis of lagging strand?
Primase- priming for Okazaki fragment
DNA polymerase- Elongation of fragment , and replacement RNA primer by DNA
Ligase- Joining of fragments
Explain the lagging strand problem(OkazakiFragments) and how is amended?
Problem:Synthesize a new primer
every few hundred bases as
more of the lagging strand is
exposed by unwinding
Result is a series of
discontinuous segments
called Okazaki fragments
* RNA primer of each fragment is
removed and replaced by DNA
solution
* DNA ligase then joins the
fragments
Describe processive synthesis
(a) DNA polymerase and the β sliding clamp are
required for processive DNA synthesis.
(b) Helicases that function at replication forks are
hexamers that encircle single-stranded DNA and
translocate on the DNA strand to separate the
strands of the parental duplex.
(c) A topoisomerase removes twists in the DNA
(d) Primase (an RNA polymerase) makes short RNA
primers to initiate DNA synthesis. Primases typically
bind the helicase, thus localizing primers to the
replication fork.
(e) Ligase seals DNA nicks, joining Okazaki
fragments together (after removal of the RNA
primers).
(f) Single-stranded DNA–binding protein (SSB) binds
cooperatively to single-stranded DNA (ssDNA),
removing secondary structure in the DNA strand and
protecting it from the action of nucleases.
DnaA
initiator bind oriC
DnaC
helicase loader
DnaB
Helicase
Gyrase
Type 2 topoisomerase A2 B2
SSB
Stabilizes and protect single strand DNA
Primase
synthesis lagging strand RNA primers
Pol 3 holoenzyme
Chromosomal replicase , consists of Pol 3 core , beta clamp , and clamp loader
Pol 1
Okazaki fragment processing : removes RNA and replaces it with DNA
Ligase
Okazaki fragment processing: joint segments
Describe the architecture of E.coli Pol 3 holoenzyme
the C-termini of the T subunit protrude from the clamp loader and bind the pol 3 cores each pol 3 core also attached to a beta sliding clamp
What are the components that make up the E.coli Pol 3 Haloenzyme?
3 pol III cores
3 beta clamps
1 clamp loader
Replicsome is made up of?
The complex of Pol 3 holoenzyme, helicase, and primase
How is helicase activity stimulated?
by its connection to the DNA polymerase, without the connection to polymerase, DnaB helicase is slow
What does DNA Replication look like in eukaryotes?
Many eukaryotic replication proteins have counterparts in bacteria( ex the clamps and clamp loader but the replication fork machinery of eukaryotes includes more proteins beyond those used in the comparatively simple bacterial machinery
new eukaryotic replicate factors are still being identified and details of the replication fork in eukaryotic cells are only now coming into focus
Important pre-replication complex proteins?
ORC and Mcm2-7
ORC
initiator
Mcm2-7
presumed helicase
Important replication complex (RC) proteins
Mcm2-7 , pol alpha , pol delta , pol epslon, PCNA , RFC , RPA
Pol Alpha
Primase
Pol Delta
Replicase in the leading strand
Pol Epsilon
Replicase in the lagging strand
What are other functions we won’t go into but exist within the replication process?
Loaders of the protein onto the DNA strand and destabilizing proteins
DNA ligase 1
Seals Okazaki fragments
Topoisomerase 1 and 2
Removes supercoil stress
What are the two different proteins involved in the leading and lagging strands seen in eukaryotes?
POL epsilon and POL delta
How are PCNA clamps connected and loaded to DNA?
the RFC clamp loader
PCNA
sliding clamp
RFC
clamp loader
RPA
single-stranded DNA binding proteins
How is an initiation of replication induced?
-Origin of replication
-the total length of DNA replicated from one Ori is called a replicon
-Binding the ORC to the ORI provides a foothold for other protein to bind and often result in strand separation in a small region of DNA at the origin
-Helicases are assembled at the unwon region paving the way for more extensive DNA unwinding and the assembly of bidirectional replication forks
What does the ORI for e.coli look like?
single origin called oriC which is 245 bp long with specific repeat sites and an AT-rich region.
Explain the order of events seen in bacteria for DNA replication.
-The assembly of bacterial replication forks at the origin occurs in steps, starting with the binding of DnaA (initiator protein), which melts an AT-rich region.
-A DnaB helicase then loaded onto each of the single strands of DNA by the DnaC helicase loader
-As DNA is unwound by DnaB, primase synthesizes RNA primers, this is followed by the entry of two POL 3 holoenzymes to form a bidirectional replication fork
Dam Methylase
Methylates adenines in GATC sequences
What is significant about hemi-methylation occurring in DNA replicaiton?
there is a protein that binds specifically to the hemimethylated DNA and blocks it from initiation of replication
Describe the difference between methlylation in prokaryotes and eukaryotes
Pro- GATC replication control
Euk-CpG gene expression / transcriptional changes
(*) is the nucleotide where the methyl group attaches
Hemi-methlyation
one of the A nucleotide in the GATC sequnces is lacking a CH3 group for a brief period of time
Describe the multiple steps that exist for the control of initiation to occur in e.coli
- Cell division requires sufficient nutrients and cell mass to support two new cells, so replication must be coordinated with the cell’s nutritional status and growth.
- Regulation occurs at the initiation step, because once replication has begun, the cell is committed to division.
- It is also of paramount importance that the origin, once replicated, can be inactivated to prevent a second round of replication during the first round, which would commit the cell to splitting twice (resulting in four cells).
- Dam methylase plays a big role in preventing this, see previous slide
What functional group must be present to allow for the replication of DNA?
available 3’ OH
What happens when the 3’OH is lacking
it leads to the problem in DNA synthesis where the chromosomes lose nucleotides during replication shorting the overall strand.
What is the mechanism that prevents this successive shortening of important material?
- Telomere repeats repeated hundreds to thousands of times. The telomeres of humans consist of as many as 2000
repeats of the sequence 5’ TTAGGG 3’. - Telomerase (RNA-protein complex) can add on telomeres
Where is the 3’OH lost in replication ?
at the end of the lagging strand the 5’ RNA is excised nothing repairs that gap and it will lead to the lost of nucleotides in the DNA strand
Explain in 4 steps how DNA replication sees the loss of nucleotides.
1.DNA replication is initiated at the origin : the replication bubble grows as the two replication fork move in opposite directions
2.Finally only one primer remains on each daughter DNA molecule
3.the last primer are removed by a 5’-3’ exonuclease but no DNA polymerase can fill the resulting gaps because there is no 3’OH available to which nucleotide can be added
4.Each round of replication generates a shorter and shorter DNA molecules
Describe what is so special about Tetrahymena what happens when you take its immortal mechanism away?
Tetrahymena, like other single-celled organisms, is immortal and can divide innumerable times. On mutagenesis of the telomerase gene, Tetrahymena telomeres shorten with each cell division, until, after 20 to 25 generations, the telomeres have shrunk below a critical level and the cells die.
Why dont humans have a immortal mechanism like tetryhymena ?
-Most somatic cells have little or no telomerase, and cells in culture divide about 40 to 60 times before losing their telomeres and dying.
-Studies in mice have shown that activation of telomerase in somatic cells leads to an increased incidence of tumors, and lifespan is shortened by cancer
Telomerase (TERT-TR)
-protein enzyme: reverse transcriptase (TERT)
-telomerase RNA (TR) ;in humans about 451 nt long
How does Tetrahymena repairs its DNA for “immortal” life span
-In Tetrahymena, reaction occurs in S phase, part of chromosome replication process
-3 nucleotides of TR anneal to 3’end of the DNA
-enzyme synthesizes single stranded DNA from RNA template
-after adding 6 nucleotide repeat, Telomerase separates the RNA-DNA hybrid and repositions on the telomere
-Once it is done, primase and polymerase and ligase take over -RNA primer is removed and now we have the same end replication issue so there is a bit of single stranded material
-shelterin proteins protects telomeres from DNA repair or chromosome joining
shelterin proteins
protects telomere from DNA repair or chromosome joining
Tetrahymena telomere are composed of numerous direct repeats give an example of one type of repeat ?
(TTGGGG)xn
What are some facts about telomerase?
- Active in germ cells and stem cells
- Turned off in most cells of adult tissue
- On in many cancer cells
When Elizabeth Blackburn what was known about replication in chromosomes?
replication machinery of the cell could not get to the very ends of the
chromosomes
Senescence
aging process where cells in culture would die
after a certain number of divisions
What was the animal model Elizabeth Blackburn used to study telomeres?
Tetrahymena: single cell ciliated protozoan
Describe a bit about the experiment Elizabeth Blackburn did to understand how Tetrahymena repair their telomeres
- analyzed the ends of the DNA using enzymes to determine the sequences that were repeated
- Made Tetrahymena extract from cells to see if there was
some enzymatic activity that could add the sequence TTGGGG over and over again to the ends of DNA right after fertilization - Made DNA oligonucleotide of GGGGTTGGGGTT… and
combined with the Tetrahymena extract+dGTP and dTTP+ Mg and found that they could be added onto the ends or the oligonucleotide - experimenters used radioactively labeled nucleotides and gel
electrophoresis to see the results of the repeating pattern—6
base pair repeat
What special mechanism did Elizabeth uncover existed in telomerase?
- they discovered that the telomerase had an RNA component,
the TR - they made changes in the RNA component and found that it
would change the DNA - there are mutations in the RNA component that block the
sequence from working entirely - Tetrahymena cells are normally immortal but if you
inactivate the RNA TR such that the telomerase enzyme no
longer works, cells start to die
What was the understanding of RNA copying before Elizabeth’s work?
- Before this work, people thought that RNA being copied
into DNA was only something that certain viruses can do but it turns out it is part of a normal cell’s life when TR is copied into telomeric DNA
Give a brief history of CRISPR
Acquired immunity in Bacteria:
Bacteria/Archea contain:
A) CRISPR locus “DNA sequences” that correspond to phage DNA sequences,
B) The Cas9 gene
Important steps in CRISPR.
Integration, expression, and interference
What is CRISPR-Cas9 in nature?
a dual RNA-guided DNA cutting
enzyme (tractRNA+CRISPR RNA).
Why are natural repair mechanisms key in cells?
they allow for disruption or insertion of DNA using donor DNA
How was CRISPR discovered? And what role might it have played in humans?
CRISPRs were later detected in numerous bacteria and archaea, and predictions were made about their possible roles in DNA repair or gene regulation
What is the original biological function? How is it a type of
immune system for bacteria?
it was proposed that CRISPR-Cas is an adaptive defense system that might use antisense RNAs as memory signatures of past invasions (. In 2007, infection experiments of the lactic acid bacterium Streptococcus thermophilus with lytic phages provided the first experimental evidence of CRISPR–Cas–mediated adaptive immunity. This finding led to the idea that natural CRISPR-Cas systems
Protein and guide RNA how does it work in CRISPR?
In 2012, the S. pyogenes CRISPR-Cas9 protein was shown to be a dual-RNA–guided DNA endonuclease that uses the tracrRNA:crRNA duplex to direct DNA cleavage. Cas9 uses its HNH domain to cleave the DNA strand that is complementary to the 20-nucleotide sequence of the crRNA; the RuvC-like domain of Cas9 cleaves the DNA strand opposite the complementary strand
What are the potential applications of CRISPR?
Research, public health, agriculture, and biomedicine
What is a big problem with CRISPR?
Due to its mechanism being a system that only induces cleavage the exact repair mechanisms are harder to mediate which can lead to off-target effects in gene therapies which can introduce a load of problems that we may not know how to solve for.
what is a replicon?
-the total length of DNA replicated from one Ori is called a replicon
