CH. 9 Genome Replication Flashcards
Viral DNA replication invariably requires at least
one viral protein, often several
Viral DNA synthesis will not begin until
there has been a sufficient accumulation of viral proteins
Replication results in
accumulation of a lot of newly synthesized molecules
In latently infected cells, replication is
strictly controlled
DNA is synthesized by stepwise incorporation of:
Deoxynucleoside monophosphates (dNMPs)
Replication of DNA begins and ends at specific sites called _____ and _____
origins; termini
DNA synthesis is catalyzed by
DNA dependent DNA polymerase
All require a ______, to which dNMP’s are added
primer with a free 3’OH
Types of virus encoded proteins
Origin binding proteins (OBP’s); helicase and primase; DNA Pol. and accessory proteins; exonucleases; nucleic acid metabolism;
Where does Pol. come from?
Small DNA virus: from the cell host; controlled by viral proteins ex. papillomaviridae, polyomaviridae, parvoviridae
Large DNA virus: can come from the virus ex. Herpesviridae, Adenoviridae, Poxviridae
Requirements for Viral DNA replication
An active cell (not quiescent)
induction of host replication enzymes and cell cycle regulators
virus encoded immediate early and early gene products
Lytic infection
new progeny
high copy number
latent infection
stable maintenance in host
Low copy number
episomal or integrated
Example of a ssDNA virus
Parvovirus (portions of the genome loop back on each other, creating some parts that are effectively double stranded
What are the two mechanisms of DNA synthesis?
Strand displacement; Replication fork
Viral origins generally have:
AT rich DNA segments that can be recognized by viral ORP. (AT bonds are weaker than CG); proximity to transcription control regions;
Current understanding of the viral DNA replication by host machinery is based on the studies of what virus?
In vitro studies of simian virus 40 (SV40)
What is a replicon?
A unit of replication. A part of a nucleic acid molecule that replicates as a unit. Contains a single origin where replication begins
How long would it take to replicate a chromosome if it was just one replicon?
10 days
What is seen in bi-directional replication
two replication forks (replication bubble) moving in opposite directions
What direction is DNA synthesized?
5’ to 3’
Why was it easy to find the origin of replication in Saccharomyces?
They support replication for just a small plasmid
Accumulating evidence suggests metazoan (animal) replication initiates within __________; what does their functioning depend on?
long DNA sequences; epigenetic (non-genetic) changes)
Where did the understanding of the replication bubble come from? What type of replication does it’s origin support?
Studying Simian Virus 40 (SV40); Bi-directional replication Its origin has a binding site for large T antigen
during SV40 replication: Which DNA Pol is responsible for priming? What is the first step?
DNA pol alpha ( it recruits primase) large T (LT) binds to the origin at pentanucleotide repeats (repeats of 5 nucleotides)
during SV40 replication: What is the second step? What are the other two proteins involved?
two hexamers of LT form, undergo conformational change, which starts to pull apart AT rich origin and early imperfect palindrome (EP). This unwinds the origin. Rp-A and cellular topoisomerase I
during SV40 replication: What is the third step?
Primase binds and adds RNA primers on the leading strand at each fork. Pol alpha extends them to make short fragments
during SV40 replication: What is the fourth step?
DNA pol alpha-primase synthesizes okazaki fragments. DNA pol Delta continues leading strand. RNA primers eliminated by exonuclease activity and RNase H
What is the purpose of Topoisomerase I and II?
They are needed to alter topology of DNA by removing supercoils to relax DNA
How does SV40 persist?
as a mini chromosome wrapped around the nucleosomes (which are histones coiled in the DNA)
SV40 replication is coordinated with what?
with the binding of new cellular DNA to cellular nucleosomes. Nucleosomes are deposited at replication forks with help of chromatin assembly factor 1
What does cellular DNA pol alpha primase complex synthesise?
RNA primers
How is adeno associated virus DNA replicated? What isn’t needed?
ssDNA forms a hairpin due to Inverted Terminal Repeats (ITR). This serves as a primer for DNA pol. Delta, Rf-C and PCNA;
Pol alpha isn’t needed.
How does Adenovirus prime?
It utilizes a protein primer complex) (Precursor to terminal protein) PTP; The displaced ss template is stabilized by DNA binding protein and ITR’s at ends
What are the common features of viral replication origins?
AT rich (2 bonds compared to 3 for CG) which facilitate unwinding; minimal essential core flanked by regions significantly increasing replication efficiency
What are the characteristics of viral origin recognition proteins?
Ability to bind specifically to DNA sequences at OOR; Bind to other replication proteins; posess biochemical activities such as helicase activity
human papillomavirus E1 possesses activity similary to:
Large T antigen of SV40 but requires additional E2 protein to replicate.
Adenovirus has the simplest viral replication apparatus, consisting of?
preterminal protein primer (PTP; DNA pol; and ss DNA binding protein
how does ss DBP work?
it multimerizes via C-Terminal hook and chains form due to high affinity for DNA
What is a DpnI sensitivity assay?
It’s an assay used to detect whether protein syn. is occuring. DNA is methylated initially, and then checked later to confirm presence of methyl groups. If they’re still there it means the DNA hasn’t been replicated.
Circular DNA replication leads to two _____ DNA molecules, requiring the use of _______
interlocked; topoisomerase II,
What is a head to tail concatamer? How is it syn.?
DNA molecule that contains multiple copies of the same sequence back to back. Synthesized by rolling circle mechanism. (herpes simplex I)
Where are Viral DNA’s synthesized for Vaccinia virus?
In the cytoplasm at discrete viral factories that contain DNA templates and replication proteins.
Nucleus synthesized DNA is generally synthesized in specialized compartments as well.
How does HPV differentiate between latency and lytic status?
It is in the epidermis. At the basal membrane the virus is in a genome maintenance stage, replicating at the same rate as the cell. As cells move apically and keratin increases, the virus enters a lytic state
