Unit 3: DNA Replication and Extrachromosomal Replication

Question 1

Topoisomerase

Answer 1

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

Question 2

Replisome

Answer 2

The multiprotein structure that assembles at the bacterial replication fork to undertake synthesis of DNA.
It contains DNA polymerase and other enzymes.

Question 3

In vitro complementation

Answer 3

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

Question 4

Replicase

Answer 4

A type of polymerase that is involved in replication. Other polymerases are involved in DNA repair or play support roles in DNA replication.

Question 5

DNA polymerase III

Answer 5

THE Bacterial replicase that makes DNA

Question 6

High-fidelity DNA polymerases

Answer 6

Involved in replication and have a precisely constrained active site that favors binding of WC base pairs.

Question 7

Processivity

Answer 7

The ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle.

Question 8

Proofreading of DNA polymerase

Answer 8

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.

Question 9

Holoenzyme

Answer 9

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.

Question 10

Leading strand

Answer 10

Daughter strand that is continuously synthesized 5’-3’

Question 11

Lagging strand

Answer 11

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.

Question 12

Semidiscontinuous replication

Answer 12

The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously.

Question 13

Helicase

Answer 13

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.

Question 14

Single-stranded protein

Answer 14

Binds to and stabilizes ssDNA being unwound by helicases. Does not unwind DNA itself.
Often found as a tetramer.

Question 15

Requirements for DNA polymerase to initiate DNA synthesis

Answer 15

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.

Question 16

E. Coli. DNA polymerase III catalytic core structure

Answer 16

Contains 3 subunits including a catalytic subunit and a proofreading subunit.

Question 17

E. Coli. DNA polymerase III holoenzyme catalytic core structures

Answer 17

Has at least two catalytic cores, a processivity clamp, and a dimerization clamp-loader complex.

Question 18

Plasmid

Answer 18

Circular, extrachromosomal DNA.

It is autonomous and can replicate itself.

Question 19

Lysogeny

Answer 19

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.

Question 20

Episome

Answer 20

A plasmid able to integrate into bacterial DNA

Question 21

Immunity

Answer 21

In plasmids, the ability of a plasmid to prevent another of the same type from becoming established in a cell.

Question 22

Strand displacement

Answer 22

A mode of replication of some viruses in which a new DNA strand grows by displacing the previous (homologous) strand of the duplex. Adenoviruses.

Question 23

Terminal protein

Answer 23

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.

Question 24

Rolling circle

Answer 24

Generates single stranded multimers of the original sequence.
Phage genomes are replicated this way.

Question 25

Conjugation

Answer 25

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.

Question 26

F Plasmid

Answer 26

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.

Question 27

Hfr

Answer 27

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.

Question 28

Copy number

Answer 28

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.

Question 29

Addiction system

Answer 29

Survival mechanism used by plasmids. Mechanism kills the bacterium upon loss of the plasmid.

Question 30

Compatibility group

Answer 30

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.

Question 31

countertranscript

Answer 31

An RNA molecule that prevents an RNA primer from initiating transcription by base pairing with the primer.

Question 32

3 Possible Primers for DNA replication

Answer 32

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

Question 33

DNA Polymerase III Holoenzyme

Answer 33

• 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.

Question 34

Plasmid

Answer 34

Circular, extrachromosomal DNA. It is autonomous and can self replicate

Question 35

Lysogeny

Answer 35

The ability of a phage to survive in a bacterium as a stable prophage component of the bacterial genome

Question 36

Episome

Answer 36

A plasmid able to integrate into bacterial DNA

Question 37

Immunity

Answer 37

In plasmids, the ability of a plasmid to prevent another of the same type from becoming established in a cell.

Question 38

Adenovirus DNA replication uses standard displacement. What is strand displacement

Answer 38

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.

Question 39

Rolling Circle DNA Replication

Answer 39

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.

Question 40

Conjugation

Answer 40

Process by which genetic material can be transferred between bacteria.
Must have an F plasmid.

Question 41

F Plasmid

Answer 41

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.

Question 42

Transfer region

Answer 42

A segment on the F plasmid that is required for bacterial conjugation

Question 43

Polycistronic

Answer 43

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.

Question 44

Pili

Answer 44

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.

Question 45

oriT

Answer 45

Origin of transfer on F plasmid.

Together with other proteins finds the relaxosome which initiates transfer into the recipient bacterium.

Question 46

What genes are more likely to be transferred during bacterial conjugation?

Answer 46

Genes closer to the oriT

Question 47

Hfr

Answer 47

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.

Question 48

Copy number

Answer 48

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

Question 49

Addiction system

Answer 49

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.

Question 50

Compatibility group

Answer 50

Comprised of plasmids that are incompatible because they have origins that are regulated by the same control system and cannot be distinguished.