Unit 3: DNA REPLICATION AND EXTRACHROMOSOMAL REPLICATION Flashcards
Topoisomerase
An enzyme that changes the number of times the two strands in a closed DNA molecule cross each other.
It does this by cutting the DNA, passing DNA through the break, and resealing the DNA
-it also Allows chromosome to be relaxed
Replisome
A multi-protein structure that assembles at the bacterial replication fork to undertake synthesis of DNA Replisomes do not exist as independent units and contain DNA polymerase and other enzymes. As the replisome moves along DNA, the parental strands unwind and daughter strands synthesize. Replisome synthesizes the daughter strand
Proteins bind to origin –> separate DNA strand –> DNAPoly and other proteins go into replisome –>synthesize daughter DNA
Conditional lethal
a mutation that is lethal under one set of conditions but is not lethal under a second set of conditions such as temperature. Conditional lethals are able to accomplish replication under permissive conditions typically provided by the normal temperature of incubation) but they are defective under non-permissive or restrictive conditions (provided by the higher temperature of 42C)
What is in vitro complementation used for?
A functional assay used to identify components of a process (in this case replication apparatus)
The reaction is reconstructed using extracts from a mutant cell aka an in vitro system for replication is prepared from a dna mutant and is operated under conditions in which the mutant gene is inactive
Fractions from wild-type cells are then tested for restoration of activity
Dna Mutants
Temperature-sensitive replication mutants that are defective in replication elongation during synthesis of DNA
Distinguish two stages of replication by their behavior when the temperature is raised
1) Quick-stop mutants
2) Slow-stop mutants
Quick-stop mutants
cease replication immediately upon a temperature increase. Defective in the components of the replication apparatus, typically in the enzyme needed for ELONGATION
Slow-stop mutants
temperature-sensitive replication mutants that are defective in INITIATION of replication. Slow-stop mutants complete the current round of replication but cannot INITIATE another round. Hence defective in initiation of new cycle of replication at the origin
DnaA-ATP and the prepriming complex bind to the origin of replication and do what?
start separating DNA (initiation of replication)
Synthesis of DNA occurs both during what two events?
-replication and DNA repair
What occurs in repair reactions?
the damage nucleotide and other bases are removed from the damaged strand and DNA polymerase comes in and synthesizes complementary strand accurately getting rid of damaged base
An enzyme that can synthesize a new DNA strand on a template strand is called what?
DNA polymerase
an enzyme that undertakes semiconservative replication (not involved in repair) is called a?
DNA replicase
Fill in the blank: DNA is synthesized by adding new nucleotides to the ____ end of the growing chain/strand
3’-OH
The incoming nucleotide that is added to the primer 3’-OH is a ? and what is released when the nucleotide is added to the growing chain?
5’ triphosphate
-diphosphate is released when nucleotide is added
DNA grows in what direction?
5’ to 3’ direction
In bacteria, which polymerase is involved in replication?
DNA polymerase III; responsible for synthesis of new strands of DNA
What is holoenzyme?
a large protein structure of replicases
What is the role of DNA poly II?
required to restart replication fork when its progressed is blocked by damage in DNA
DNA polymerase IV and DNA polymerase IV are both involved in what?
translesion replication. Involved in allowing replication to bypass certain types of damage and are called error-prone polymerases
What is meant by semiconservative replication?
The two strand of parental duplex are separated and each serve as a template for synthesis of daughter strand. Thus, there will be two daughter duplexes, each of which has one parental strand and one newly synthesized strand
What is a DNA repair reaction?
Repair synthesis replaces a short stretch of one strand of DNA containing a damaged base.
One strand of DNA is damaged. It is excised and new material is synthesized to replace it.
All prokaryotic and eukaryotic DNA polymerases share the same fundamental type of synthesis activity. What is it?
- antiparallel synthesis from 5’ to 3’ from a template that is 3’ to 5’
- this means adding nucleotides one at a time to a 3’-hydroxyl (OH) growing end meaning chain grows in the 5’-3’ direction
- the choice of the nucleotide to add to the chain is dictated by base pairing with the complementary template strand
What is the unique role of DNA polymerase I?
DNA poly I has a unique 5’-3’ exonuclease activity. The exonuclease activity is also used to excise bases that have been added to DNA incorrectly. This provides a proofreading error control system. This exonuclease activity can be combined with DNA synthesis to perform nick translation.
What happens to DNA pol I when treated with protease? What are the subunits of Pol I?
DNA pol I cleaves into two subunits when treated w/a protease. The large subunit is called the cleanout fragment and the smallest subunit has the 5’-3’ exonuclease activity which removes about 10 bases at a time.
What is a nick translation? when does it occur?
nick translation replaces part of a preexisting strand of duplex DNA w/newly synthesized material. Nick generates 3’-OH, 5’-P groups.
Nick translation occurs when there is a nick on one strand of the DNA
What degrades the old strand replaced by nick translation when there is a nick on one strand of DNA?
The old strand gets degraded by the 5’-3’ exonuclease activity while the cleanout fragment synthesizes a new strand as a result the nick gets translated along the DNA strand.
What is meant by high fidelity DNA polymerases?
These polymerases are involved in replication that have a precisely constrained active site that favors binding of Watson-Crick base pairs. These polymerases are sensitive to change of structure and they use the geometry of the nucleotide pair to determine if the pairing is accurate
-if the pairing is mismatched it is unlikely to fit into the active site.
What is meant by low fidelity polymerases?
Low fidelity polymerases are most often used for repair. They have a more open active site and can tolerate not only damaged based pairs but also mismatch pairs
examples: e.coli DNA poly IV that is used for damage bypass replication.
poly IV have open active sites that accommodates damaged nucleotides, but also incorrect base pairs.
What is meant by processivity?
Processivity of enzyme is the ability of the enzyme to catalyze multiple reaction cycles on a single template without falling off between cycles and then getting back on.
aka the tendency to remain on a single template rather than dissociate and reassociate
How do DNA polymerases control the fidelity of replication?
bacterial polymerase have a 5’-3’ exonuclease activity that is used to excise incorrectly paired bases and proofread which controls the fidelity of replication. DNA polymerases will check the nucleotide added at the end of the growing chain and will remove it if the nucleotide is incorrect.
Proofreading by DNA poly improves fidelity 100 fold
How does DNA polymerase know that an incorrect base has been incorporated?
If an incorrect base is added then DNA helical structure gets warped and this will cause the DNA polymerase to slow down. This slowing down is a sign that the DNA 5’-3’ exonuclease activity needs to be exercised
What is the advantage of a holoenzyme as opposed to a single unit?
The advantage of holoenzyme is that you can have the exonuclease part of the enzyme in a different subunit and this increases fidelity of a repair enzyme
Explain how polymerase proofread:
1) Enzyme adds base to growing strand (3’-OH growing strand)
2) Enzyme moves on if new base is correct
3) Base is hydrolyzed and expelled if incorrect
What is frameshift error?
occurs when an extra nucleotide is inserted or omitted. Fidelity w/regard to frame-shifts is affected by the processivity of the enzyme
What is the klenow fragment
DNA polymerase I, the first DNA polymerase for which the structure was determined. Characterized by 3 domains: palm, fingers, and thumb (resembles a hand)
In the Klenow structure where does the DNA sit?
across the palm, (groove cleft), between the finger and the thumb.
The palm domain has important conserved sequence motif that provide the catalytic site
In the Klenow structure what is the role of the finger?
involved in positioning the DNA template correctly at the active site.
In the klenow structure what is the role of the thumb?
the thumb binds the DNA as it leaves the enzyme and is key for processivity of the enzyme.
What is semidiscontinous replication?
The mode of replication in which one strand is synthesized continuously (leading strand 5’-3’), while the other is synthesized discontinuously (lagging strand is synthesized by making short okazaki fragments that are later joined together)
What elements are needed to convert DNA from double to single strand?
1) helicase
2) single-strand binding protein (SSB)
Replication requires a helicase to separate the strands of DNA using energy provided by hydrolysis of ATP
A single stranded DNA binding protein is required to maintain the separated strand
What is the structure of a helixase?
multi-meric wi/hexamer being the most common form.
What is the structure of SSB?
a tetramer
How does SSB bind with the DNA?
cooperatively preventing the duplex from reforming
Why does the antiparallel structure of the two strands of duplex DNA pose a problem for replication?
- As the replication fork advances, daughter strands must be synthesized on both of the exposed parental strands
- The fork template strands moves in the direction from 5’-3’ on one strand and in 3’-5’ on the other strand.
YET!!! DNA is synthesized only from a 5’ end toward a 3’ end (by adding a new nucleotide to the growing 3’ end)
How is the antiparallel structure of DNA issue resolved when replicating DNA?
The problem is solved by synthesizing the new strand on the 5’ to 3’ template in a series of short fragments, each synthesized in the ‘backward’ direction; That is with the customary 5’-3’ polarity
What is required for DNA polymerase to start DNA synthesis?
pre-existing 3’-OH end (PRIMER) to initiate synthesis. The template strand must also be converted to a single strand.
TRUE OR FALSE: DNA polymerase can start DNA synthesis on their own
FALSE: DNA polymerases cannot start synthesizing the DNA strand on their own. They need a preexisting 3’-OH hydroxyl and it can then elongate by adding nucleotides to the 3’-hydroxyl group
How can the priming end or primer be provided to a DNA strand?
There are several methods for providing the free 3’-OH end that DNA polymerases require to initiate DNA synthesis
1) RNA primer: is synthesized by a primase (or provided by base pairing RNA → DNA)
2) A nick in DNA: Duplex DNA is nicked to provide free end for DNA polymerase
3) Priming protein: A priming nucleotide is provided by a protein that binds to DNA
How many primers are required on the leading and lagging strand for DNA polymerase to initiate synthesis?
The leading strand only requires one such primer while the lagging requires several such primers since each okazaki fragment requires its own primer.
Each okazaki fragment begins with a RNA primer of about 10 bases long
Each okazaki fragment begins with a RNA primer of about 10 bases long. Where does the RNA primer come from?
from a special RNA polymerase called a primase.
e.coli has two types of priming reactions which occur at the bacterial origin (oriC) and the phageX174
What is the differences between the two priming reactions for e.coli (oriC vs. phageX174)?
ori used DnaG primase to make its primer while phageX173 requires primosome to synthesize a primer and this is used when damage causes the replication fork to collapse and needs to be restarted
Different enzymes units are required to synthesize the leading and lagging strands. Why?
Because leading and lagging are moving in different directions. The unit moving along the leading strand is moving in the same direction as the replication fork. The unit in the lagging strand is moving in a sense ‘backwards’
Each okazaki fragment has to be completely synthesized before the next fragment can begin synthesis at the replication fork. This means the DNA polymerase has to disengage from the template, move to the new location, and reconnect to the template and using the next primer start the next okazaki fragment
In e.coli the catalytic subunit is DnaE
In other like eukaryotes there are different catalytic subunits for each strand
DnaE is?
the catalytic subunit in e.coli required to synthesize the leading and lagging strand.
The DNA polymerase holoenzyme (pol III) consists of subcomplexes, what are they?
The replisome in e.coli consists of DNA pol III holoenzyme complex in association w/a primase and a helicase. The DNA pol III core contains 3 subunits including the catalytic subunit and proofreading subunit
What are the catalytic cores of DNA polymerase III holoenzyme?
The holoenzyme has at least two catalytic cores: the processivity clamp and dimerization clamp loader complex
It is proposed that DNA pol III has 3 polymerases cores, what are they?
two catalytic cores responsible for synthesis of the lagging strand and one catalytic core responsible for the leading strand
Each okazaki strand is synthesized by the alternating core polymerase
What is the role of a clamp loader?
The clamp loader is a complex of seven proteins and its function is to place the processivity units (the beta subunits) on DNA to form the beta ring. The clamp is responsible for holding the catalytic core on to the template strands
-forms a circular clamp around the nucleic acid
What is the catalytic core made up of?
The catalytic core is made up of the alpha subunit, which has the polymerase activity, epsilon - which has the 3’-5’ proofreading activity, and the theta subunit which stimulates the exonuclease
How does DNA polymerase III assemble? (short explanation)
in stages, generating an enzyme complex that synthesizes
1) Clamp loader cleaves ATP to load clamp on DNA
2) Core enzyme joins
3) Tau + second core joins to give a symmetric dimmer
4) Tau subunits maintain dimeric structure
long explanation for assembly of DNA poly III
The clamp loader uses the energy from the hydrolysis of ATP to bind the beta units to the template primer complex. Once the beta subunits bind to the DNA it is thought that the beta subunits have now increased affinity for the core complex through a conformational change, thus recruiting the core complex to the replication site
The tau dimer which is part of the clamp loader links to a second core complex and helps to maintain the dimeric structure
Each of the catalytic cores synthesizes one of the new strands of DNA namely the leading strand and the lagging strand
Is the clamp on leading strand processive? What about lagging?
Yes, the clamp on leading is processive b/c its clamp keeps it on the DNA
On the lagging strand, the clamps is not processive b/c it dissociates at the end of each okazaki fragment and reassembles for the next fragment
Why does the lagging strand create a loop, which gets bigger as the undwining of DNA advances?
as the holoenzyme moves continuously to form the leading strand the lagging strand gets pulled through creating a loop which gets bigger as the unwinding advances
The lagging strand core complex pulls the second single stranded template through the strand while synthesizing the new strand
The template should extend the length of at least one okazaki fragment before the polymerase on the lagging strand can start synthesizing the next fragment
What is the role of helicase DnaB? okazaki
responsible for interacting w/the primase DNAG to initiate each Okazaki fragment.. Forward movement of the fork
What happens to the clamp once an okazaki fragment is completely synthesized
Once okazaki is completely synthesized it will require the complete dissociation of the clamp from core poly and the reassociate again. The clamp loader causes the beta clamp to open up by changing its conformation
A new beta clamp is then recruited by the clamp loader to start the synthesis of the next okazaki fragment
STEPS
1) initiation of okazaki fragment
2) termination of okazaki fragment
3) beta clamp dissociates
4) beta clamp associates
TRUE OR FALSE: Each okazaki fragment starts w/a primer and stops before the next fragment
TRUE; DNA polymerase I removes the primer and replaces it with DNA
What is the role of polymerase I in okazaki fragments?
Primer is RNA primer and needs to be removed and replaced with DNA, this is done by polymerase I using nick translation
What steps are involved in synthesis of okazaki fragments? prokarotes
Require priming, extension, removal of RNA primer, gap filling, and nick ligation
1) Primase synthesizes RNA
2) DNA Pol III extends RNA primer into okazaki fragments
3) Next Okazaki fragment is synthesized
4) DNA polymerase I uses nick translation to replace RNA primer w/DNA
5) Ligase seals the nick
How are okazaki fragments linked?
by ligase;
DNA ligase makes the bond that connects the 3’ end of one Okazaki fragment to the 5’ beginning of the next fragment
DNA ligase seals nick between adjacent nucleotides by employing an enzyme-AMP intermediate
What happens when there’s damage to DNA?
If it’s not repaired it can be lethal so it bypasses lesion.
The replication fork stalls when it arrives at the damaged site and may collapse. This has to be replaced by a specialized DNA polymerase that can replicate through the lesion.
In e.coli two error prone polymerases IV and V and pol II are used for the translesion synthesis
What happens after damage DNA has been bypassed by Poly IV and V
Primosome reinserts the replication complex after polymerase IV and V have repaired damage
-It reloads the DnaB so that the helicase activity can resume
THUS, the primosome is required to reinitiate replication or restart stalled replication fork by reinserting the replication complex
How does replication terminate?
The two replication forks usually meet halfway around the circle, but there are ter sites that cause termination if the replication forks go too far.
These sites are situated near termination region
Each set of ter fork allow the replication fork to the termination region but does not allow it out the other side
So replication fork one can pass through ter B and ter C but cannot go to ter A, D or E
This serves as a replication fork trap. Hence if one replication fork is moving slower than the other the replication fork trapp will effectively trap the faster moving fork at the ter site. Until fork two catches up
Plasmids
circular, extrachromosomal DNA. It is autonomous and can replicate itself
Lysogeny
the ability of a phage to survive in bacteria as a stable prophage component of the bacterial genome
Episome
if plasmid can integrate into the bacterial DNA/chromosome it is called an episome
Immunity
plasmids and lysogenic phages do not allow another element of the same type to become established in the bacteria or cell -this ability is called immunity
Why are the ends of linear DNA a in bacteria a problem for replication?
if we have linear DNA then the DNA polymerase needs to initiate replication at the 5’ end since the progression of the newly formed strand is in the 5’ to 3’ direction. There has to be some way for replication machinery to start at the very end of the template strand. it could do that by either converting the linear form into a circular or multimeric form as in T4, or the Lambda phage, or it could form long hairpin structures in the end as seen in paramecium. or it could use proteins which bind to the 5’ end as seen in adenoviruses
TRUE OR FALSE:
Terminal proteins enable initiation at the ends of viral DNAs
TRUE; example adenovirus
What is strand displacement?
Strand displacement - a mode of replication of some viruses in which a new DNA strand grows by displacing the previous (homologous) strand of the duplex.
Adenovirus DNA replication is initiated separately at the two ends of the molecule and proceeds by strand displacement
In virus that use strand displacement, a terminal protein binds to the 5’ end of DNA, what is the role of this?
1) provides cytidine nucleotide with a 3’-OH group, which serves to prime replication.
2) terminal protein also associates DNA polymerase. the complex of the terminal protein and DNA polymerase bind to the5’ end of the DNA. elongation of the newly synthesized strand by the polymerase will result in a new strand that is bound to the initiating C nucleotide. the old terminal protein gets displaced
What is the rolling circle method?
-a rolling circle generate single-stranded multimers of the original sequence.
in the rolling circle method only one strand is used to generate several copies of the original sequence
if the template is a circular duplex DNA., one of the strands undergoes a Nick so providing a free 3’ hydroxyl group for the DNA polymerase to come in and synthesizes the complementary strand. as the new strand elongates it displaces the old strand generating a single stranded tail. now the polymerase can keep going around the circle indefinitely performing a multimeric single stranded tail with the original unit at the 5’ end.
What determines the types of products generated by rolling circles?
The fate of the displaced tail
The displaced tails could get cleaved at unit lengths generating monomers or it could be cleaned as multiples which can remain linear or it could form a duplex by the synthesis of the complementary strand it could circularize to form single strand and duplex regular forms
What is the phage. A protein?
a cis-acting relaxase that generates single-stranded circles from the tail produced by rolling circle replication.
phageX174 DNA is a template for synthesizing single-stranded viral circles. What are the steps?
we see rolling circles when phage genomes are replicating.
1) PhageX174 genome is a single stranded circular DNA molecule and is referred to as the plus strand. for replication first the strand has to be converted into a duplex circle by synthesizing a complementary strand referred to as the minus strand.
2) the replication now proceeds in the rolling circle format.
3) the A protein nicks the positive strand and stays bound to the 5’ end of the strand, while the 3’ end gets extended by the polymerase. as it completes the circle it displaces the original strand
4) the A protein which is still bound to the 5’ end is still associated w/the rolling circle and will recognize the origin of the next rolling circle and nick it again, attaching itself to the 5’ end of the newly synthesized circle so that the next round of the low rolling circle can begin
What is congjuation?
A process in which two cells come in contact and transfer genetic material. This process requires the presence of F plasmid
What is an F plasmid?
The free F plasmid is a large circular DNA and a replicon that is maintained at the level of one plasmid per bacterial chromosome
-An F plasmid is an episome and can exist as a free plasmid or can integrate into the bacterial chromosome, in which case its own replication system is suppressed. Since it has its own origin of replication, which it utilizes if it is in the free form, it is maintained as a single copy per cell
What is the transfer region on f plasmid
A segment of an F plasmid that is required for bacterial conjugation
What happens to the ori on an F plasmid, if the F plasmid is integrated into the bacterial genome?
then the F-plasmid will not use its own Ori for replication but rather gets replicated as part of the bacterial genome.
What is found at the regulatory region of plasmid?
a TraJ gene is found in this region
What is the role of TraJ?
it turns on both TraM and TreI/Y
What is the role of the regulator FinP?
It turns off TraJ
How many Tra and Trb genes are involved directly with transfer of DNA?
only four
Pili
form on the surface of the bacterium and are encoded by F plasmids
Pilin
coded by gene TraA and polymerizes to form the pilus which is a hollow cylinder
What role do pilus play on F positive cells?
F positive have 2-3 pilus and enables an F-positive bacterium to contact/connect to an F-negative bacterium and initiate conjugation
TraS and TraT code for what on the surface of proteins?
code for surface exclusion proteins that prevent an F-positive cell from meeting w/another F-positive cell. After initial pairing of these cells, the F pili retract and disassmeble
Transfer of DNA between F positive and F negative is provided by what ?
through a channel provided by the T4SS which is a membrane spanning protein complex
Addiction system
a survival mechanisms produced by plasmids where the mechanism kills bacterium upon loss of plasmid.
Plasmid produces long lived killer substance and short lived antidote substance. plasmid provides antidote. If loss of plasmid occurs antidote will stop being produced and killer will kill bacterium
