Lectures 5 & 6- Genome Replication - DNA viruses

Question 1

Enzymes for replication and transcription are called by different names

Answer 1

DNA viruses: replication = dna polymerases, transcription = rna polymerases
RNA viruses: replication = replicase (RNA polymerase) or reverse transcriptase (retrovirus), transcription = transcriptase (RNA polymerase)

Question 2

Some viruses code for their own DNA polymerase while others use host enzymes- which ones?

Answer 2

Large DNA viruses code for own DNA polymerases. Similar to DNA pol that cells produce. Examples: T4, T7, adenoviruses, herpesviruses, poxviruses
Small DNA viruses use host DNA synthesis machinery. Not a lot of genetic capacity. Examples: PhiX174, M13, Lambda, SV40, papilloma viruses
RNA viruses (retroviruses are a special case) code for own replicases and transcriptases. They require RNA pol and almost exclusively replicate in cytosol of euk cells

Question 3

General properties of DNA polymerases

Answer 3

1) Template strand instructs the DNA polymerase regarding the order of nucleotides in new chain.
2) DNA strands are synthesized by the successive addition of nucleotides onto the 3’ end of the nascent chain
3) DNA polymerases require a primer (usually RNA) to initiate a new chains
Properties of DNA pol lead to end problem

Question 4

Prok vs euk DNA replication

Answer 4

Prok:
circular, ori c location
bi directional from single origin
topoisomerases present
called theta mode of replication
no viruses that replicate by same mode as euk
Euk: ds dna (linear) forks converge: replication completed

Question 5

The end problem in DNA replication

Answer 5

Remove RNA primer on lagging strand: newly synthesized chains with 5’ end cannot be completed

Question 6

Viral Solutions to the end problem

Answer 6

A: replicate as circles
B: Form concatemers
C: Use of special DNA ends
D: Use of “protein” primers

Question 7

Replicate as circles

Answer 7

No ends no problem
1) Genome = ds circular DNA. Examples: polyoma, SV40, papilloma virus
2) Genome is ss circular DNA (ssDNA converted to dsDNA). Examples: phiX174, filamentous phages fd and M13. ss almost always converted to ds by cell machinery
3) Linear genome circularizes
Examples: lambda phage circularizes using cohesive ends and herpes viruses circularize by blunt end ligation

Question 8

Phage lambda solution to the end problem

Answer 8

circularizes using cohesive ends that are 12 nucleotides long
5’ ss extensions that are 12 nucleotides long
complementary sequences cohere by way of “cohesive ends”
stick together through BPing
spontaneous BPing reaction, occurs quickly after infection
ds circular molecule with both strands intact
Ligase seals nicks
Replicates by theta mode (then gets more complicated)

Question 9

Form concatemers

Answer 9

A concatemer is made up of end to end tandem repeats of the genome
4 genomes in end to end array
Bacteriophage T7 uses terminal redundancy to form concatemers during replication (continuous polynucleotide chains)
concatemer of molecule, can be 100’s of genomes in length

Question 10

Bacteriophage T7 replication solution to end problem

Answer 10

form concatemers
two termini are the same (redundant sequences)
both 5' ends are incomplete
they base pair
dna ligase seals up breaks
like the simplest concatemer

Question 11

Use of special DNA ends

Answer 11

Displacement vs non displacement synthesis:
Non displacement: nascent chain does not have to displace non template strand
Displacement: usually requires some DNA pol and helices, some DNA pol can act alone. Does displace non template strand.

Question 12

Use of special DNA ends- parvovirus

Answer 12

Parvovirus genomes are single stranded and both polarities. They have hairpin ends. There is a special sequence at the end of genome- enables hairpin structure to form.
Parvoviruses have hairpin ends that are self-priming. End of parental dana will fold on itself (form hairpin structure) and once the 3’ end is bped, it can extend
Get sequence specific endonuclease cleaveage between folded part and bottom strand. Then get displacement synthesis. Ss progeny DNA on top, parental on bottom
Intermediate: one of the products can be recycled back to seq specific endonuclease cleavage step. Top strand is displaced (new), bottom is extended (template). Get ss progeny DNA, parental DNA recycled

Question 13

Use of special DNA ends- poxvirus

Answer 13

The continuous ends on poxvirus DNA solve the end problem
very large ds DNA with weird ends
both ends are continuous, which is atypical
seq specific cleavage occurs (will cleave both c’ locations), followed by displacement synthesis

Question 14

Protein acts as a primer (which viruses?)

Answer 14

examples: adenoviruses, phage phi29, hep B, and poliovirus

Question 15

Adenovirus use of protein primers

Answer 15

Adenoviruses initiate synthesis from a “protein” primer
ds linear molecule, terminal protein-ser-phos-c’ at each 5’ end (covalent linkage between TP and 5’)
this is leftover from when the molecule was produced, not relevant in the next cycle of replication.
1) Pol bout dot terminal protein, C residue added to Pol-TP, bp to G in template strand. C residue can now be extended. Top strand displaced, now primer generates new ds molecule by displacement synthesis.
2) Displaced strand plus another DNA pol-TP-dCTP will give a second ds DNA molecule

Question 16

Processing of concatemers to produce unit length genomes

Answer 16

concatemers are produced by
1) end joining of linear during phage T7 replication
2) rolling circle replication
Rolling circle replication (phiX174, M13, lambda, and herpes viruses) (lambda and herpes viruses are linear and circularize)

Question 17

Production of phage lambda genomes from concatemers

Answer 17

Concatemer is cleaved by site-specific endonuclease to produce staggered cuts 12 bp apart. The site of cleavage is called cos and the enzyme is referred to as the terminate. Resultant genomes have cohesive ends.

Question 18

How is concatemer cleavage coupled to assembly?

Answer 18

Start with empty capsid with opening, where DNA threaded into empty head to be packaged/spooled inside structure
Opening has DNA translocating vertex (portal vertex). Terminate cleaves at cos site. Terminate sites at edge and cleavers as DNA is threaded.
Cutting of concatemer is linked to packaging

Question 19

DNA packaging by bacteriophage phi29

Answer 19

Big takeaway is FORCE
PE to KE: when being injected into cell, driving force for shooting DNA into cell is the tight packaging
DNA being spiralized inside
Machinery threading DNA is like an engine (ATPase). 1 ATP hydrolyzed for every 2 bp packaged
Takes a lot of energy; DNA captured under great pressure (60 atm- champagne bottle)
Tail added after; helps inject

Question 20

Production of circularly-permuted terminally redundant T7 genomes from concatemers (260 bp)

Answer 20

Cleave so that you get ABC at both ends of a strand
Get genomes where first begins and ends with A, second begins and ends with B, etc.
Still terminally redundant, but not the same as original
Do not start with the circle; start with concatemer
T4’s replicate through concatemeric structure
Since ends are different, cleavage must not be site-specific.

Question 21

In production of circularly-permuted and terminally-redundant T4 genomes from concatemers, when does terminase cleave?

Answer 21

Not site specific b/c genomes not all identical. Uses the “headful” packaging mechanism. Terminate cleaves when head is full. This allows for one redundancy, whichever one it happens to be is okay. Does not cleave at a sequence, cleaves when head is full.