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
Replisome
The multiprotein structure that assembles at the bacterial replication fork to undertake synthesis of DNA.
It contains DNA polymerase and other enzymes.
In vitro complementation
A functional assay used to identify components of a process.
- The reaction is reconstructed using extracts from a mutant cell
- Fractions from wild-type cells are then tested for restoration of activity
Replicase
A type of polymerase that is involved in replication. Other polymerases are involved in DNA repair or play support roles in DNA replication.
DNA polymerase III
THE Bacterial replicase that makes DNA
High-fidelity DNA polymerases
Involved in replication and have a precisely constrained active site that favors binding of WC base pairs.
Processivity
The ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle.
Proofreading of DNA polymerase
DNA polymerases often have a 3’-5’ exonuclease activity that is used to excise incorrectly paired bases.
The fidelity of replication is improved by proofreading by a factor of ~100.
Holoenzyme
A bunch of enzymes or enzymes and cofactors that are part of their own larger complex. DNA polymerase involved in replication is an example of this. DNA polymerases involved in repair are not.
Leading strand
Daughter strand that is continuously synthesized 5’-3’
Lagging strand
Daughter strand that is synthesized in short fragments because its template is 5’-3’ (and the polymerase is moving 3’-5’). Short fragments are called Okasaki fragments.
Semidiscontinuous replication
The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously.
Helicase
Helicases are enzymes that utilize the energy derived from adenosine triphosphate (ATP) hydrolysis to separate the complementary strands of a DNA or RNA duplex.
Generally multimeric - hexamer is common form.
Unwinds several kilobases at a time, therefore needs a single-stranded protein to bind and stabilize ssDNA.
Single-stranded protein
Binds to and stabilizes ssDNA being unwound by helicases. Does not unwind DNA itself.
Often found as a tetramer.
Requirements for DNA polymerase to initiate DNA synthesis
DNA polymerases cannot initiate DNA synthesis on their own.
DNA polymerases require a 3’-OH to initiate replication.
Priming is required to start synthesis which can be provided by: an RNA primer, a nick in the DNA, or a priming protein. All are methods for proving a 3’-OH.
Leading strand only needs one primer.
Lagging strand requires a primer for each Okasaki fragment.
E. Coli. DNA polymerase III catalytic core structure
Contains 3 subunits including a catalytic subunit and a proofreading subunit.
E. Coli. DNA polymerase III holoenzyme catalytic core structures
Has at least two catalytic cores, a processivity clamp, and a dimerization clamp-loader complex.
Plasmid
Circular, extrachromosomal DNA.
It is autonomous and can replicate itself.
Lysogeny
The ability of a phage to survive in a bacterium as a stable prophage component of the bacterial genome.
Phages DNA has integrated into the bacterial genome.
Episome
A plasmid able to integrate into bacterial DNA
Immunity
In plasmids, the ability of a plasmid to prevent another of the same type from becoming established in a cell.
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. Adenoviruses.
Terminal protein
Binds to the 5’ end of DNA and provides a cytidine and nucleotide with a 3’-OH end that primes replication.
Also associates with DNA polymerase.
Rolling circle
Generates single stranded multimers of the original sequence.
Phage genomes are replicated this way.
Conjugation
A process in which two cells come in contact and transfer genetic material.
In bacteria, DNA is transferred from a donor to a recipient cell.
F-plasmid is transferred by conjugation between bacteria.
F Plasmid
Is a large circular DNA of about 100 kb. Is an “episome”, that is, it can exist as a free plasmid or be integrated into the bacterial genome. If existing on its own, has its own origin for replication. The “transfer region” is a segment on the F plasmid that is required for bacterial conjugation.
Hfr
A bacterium that has an integrated F plasmid with its chromosome.
Hfr = high frequency recombination = referrs to the fact that chromosomal genes are transferred from an Hfr cell to an F- cell much more frequently than from a F+ cell.
Copy number
The number of copies of a plasmid that is maintained in a bacterium (relative to the number of copies of the origin of the bacterial chromosome)
Single-copy plasmids exist at one plasmid copy per bacterial chromosome origin.
Multicopy plasmid exist at >1 plasmid copy per bacterial chromosome origin. Generally between 10 and 20. Several initiation events per cell cycle.
Addiction system
Survival mechanism used by plasmids. Mechanism kills the bacterium upon loss of the plasmid.
Compatibility group
Plasmids in a single group have origins that are regulated by a common control system.
If plasmids belong to the same compatibility group their origins cannot be distinguished at initiation and are imcompatible.
countertranscript
An RNA molecule that prevents an RNA primer from initiating transcription by base pairing with the primer.
3 Possible Primers for DNA replication
DNA polymerases require a 3’-OH priming end to initiate DNA synthesis
Primer can be:
1) RNA fragment - RNA primer is synthesized by RNA primase or provided by base pairing
2) Nick in DNA - Duplex DNA is nicked to provide a free end for DNA polymerase
3) primer protein - A priming nucleotide is provided by a protein that binds to DNA
DNA Polymerase III Holoenzyme
- 2 DNA Pol III enzymes, each comprising α, ε and θ subunits. (It has been proven that there is a third copy of Pol III at the replisome.[1])
- the α subunit (encoded by the dnaE gene) has the polymerase activity.
- the ε subunit (dnaQ) has 3’→5’ exonuclease activity.
- the θ subunit (holE) stimulates the ε subunit’s proofreading. - 2 β units (dnaN) which act as sliding DNA clamps, they keep the polymerase bound to the DNA.
- 2 τ units (dnaX) which act to dimerize two of the core enzymes (α, ε, and θ subunits).
- 1 γ unit (also dnaX) which acts as a clamp loader for the lagging strand Okazaki fragments, helping the two β subunits to form a unit and bind to DNA. The γ unit is made up of 5 γ subunits which include 3 γ subunits, 1 δ subunit (holA), and 1 δ’ subunit (holB). The δ is involved in copying of the lagging strand.
- Χ (holC) and Ψ (holD) which form a 1:1 complex and bind to γ or τ. X can also mediate the switch from RNA primer to DNA.
Plasmid
Circular, extrachromosomal DNA. It is autonomous and can self replicate
Lysogeny
The ability of a phage to survive in a bacterium as a stable prophage component of the bacterial genome
Episome
A plasmid able to integrate into bacterial DNA
Immunity
In plasmids, the ability of a plasmid to prevent another of the same type from becoming established in a cell.
Adenovirus DNA replication uses standard displacement. What is strand displacement
Strand displacement - A mode of DNA replication of some viruses in which a new DNA strand grows by displacing the previous (homologous) strand of the duplex.
Rolling Circle DNA Replication
A rolling circle generates single stranded multimers of the original sequence.
One strand undergoes a nick which provides a 3’-OH. As strand elongates it replaces the original strand..
Long tail is formed and could form multimeric strands and various other structures.
Conjugation
Process by which genetic material can be transferred between bacteria.
Must have an F plasmid.
F Plasmid
A replicon that is maintained at the level of one plasmid per bacterial chromosome. Can integrate into the bacterial chromosome, in which case its own replication system is suppressed.
Transfer region
A segment on the F plasmid that is required for bacterial conjugation
Polycistronic
The term “polycistronic” describes the situation in which two (bicistronic/dicistronic), three (tricistronic), or more separate proteins are encoded on a single molecule of messenger RNA (mRNA). In prokaryotes, polycistronic expression is common.
Pili
Hairlike structures that form on the surface of bacterium.
Transfers F plasmid genetic material between bacteria. Must be transferred from F-positive bacterium to a F-negative bacterium to initiate conjugation.
oriT
Origin of transfer on F plasmid.
Together with other proteins finds the relaxosome which initiates transfer into the recipient bacterium.
What genes are more likely to be transferred during bacterial conjugation?
Genes closer to the oriT
Hfr
A bacterium that has a high frequency of recombination. Has integrated F plasmid with its chromosome.
Genes are transferred from an Hfr cell to an F- cell much more frequently than from an F+ cell.
Copy number
The number of copies of a plasmid that is maintained in a bacterium (relative to the number of copies of the origin on the bacterial chromosome)
Single-copy plasmids exist at one plasmid copy per bacterial chromosome origin
Addiction system
A survival mechanism used by plasmids.
Plasmid makes long live toxin and short-lived antidote. If it loses the plasmid, the toxin lives longer than the antidote and the bacterial cell dies.
Compatibility group
Comprised of plasmids that are incompatible because they have origins that are regulated by the same control system and cannot be distinguished.
