Lecture 2/5 Flashcards
In which direction does replication take place in the cell?
It is bidirectional, with a replication fork on each side of the replication bubble
Do prokaryotes have a cell cycle?
No
What is the function of topoisomerase?
It relaxes the supercoils of DNA (cuts through) made by helicase
Where does topoisomerase cleave DNA? And does it put the DNA back together after?
At the phosphate backbone, and it ligates the strands back together afterward
Where do exonucleases work?
At a free end
Is topoisomerase an exo or endonuclease?
Endonuclease, it also ligates.
What is the order of proteins recruited for DNA replication in prokaryotes?
Initiator protein binds to the origin of replication, recruits helicase to unwind, ssBPs to keep the replication bubble open, primase to make an RNA primer, DNA pol iii for elongation, primers removed by DNA pol i and fragments put together by ligase.
How does DNA polymerase elongate on both strands of DNA concurrently when the direction of elongation is in opposite directions?
The DNA polymerase complex twists the lagging strand
Where are nucleotides removed from in proofreading?
The 3’ end
If there is a mismatch during elongation, the DNA is destabilized in the polymerase active site. How is it fixed?
The DNA is transferred to an exonuclease site, the mismatch is corrected and then flipped back to the polymerase active site
If a DNA mismatch is not initially caught by DNA polymerase, can it still be fixed?
Yes, but it will be fixed later and not by polymerase
In prokaryotes, does DNA replicate from one site or multiple sites? And is it random?
One site of origin, not random
Which is faster, pol i or pol iii?
Pol iii is much faster
When is replication finished in a prokaryote?
when the replication forks meet at the termination region and the topoisomerases separate the entwined chromosomes, yielding 2 molecules
What is needed for DNA synthesis in a tube?
dNTPs, Mg++ (co-factor), DNA template, DNA primer, polymerase
are all 3 DNA polymerases capable of elongation?
yes, in the 5’ to 3’ direction
are all 3 DNA polymerases capable of 3’ to 5’ exonuclease activity?
yes
what is 3’ to 5’ exonuclease activity?
proofreading
are all 3 DNA polymerases capable of 5’ to 3’ exonuclease activity?
no, only pol i
what is 5’ to 3’ exonuclease activity?
removal of RNA primers
what are the 5 major differences between DNA replication in euks an proks
- many replication bubbles
- primase works in complex with DNA pol alpha; elongation with DNA pol delta + PCNA + RF-C (lag) and epsilon + helicase (lead)
- RNA primers degraded by ribonucleases HI and FEN-1
- Histone proteins must be dissociated from DNA
- DNA is ds linear
Why do euks have multiple replication bubbles?
the genome is much larger, and replication is much slower than in proks
what is the origin site called in euks?
ORC - origin of replication complex
how long does it take bacteria to replicate?
20 mins
what does primase + dna pol alpha do?
synthesizes the primer
which polymerases + proteins are used in elongation in euks?
leading strand: dna pol epsilon + helicase
lagging strand: dna pol delta + PCNA + RF-C
which replicates faster in euks, leading or lagging strand?
they are the same speed
what is used in eukaryotes to unwind and keep the replication bubble open?
helicase unwinds, SSBPs keep it open, and topoisomerases cut the supercoiled DNA and ligate it after to re-seal
What does RF-C do?
It binds new DNA strand and recruits PCNA to help load DNA onto polymerases
What switch is required for elongation in euks?
the switch from DNA pol alpha to either pol delta (lagging) or pol epsilon (leading)
what degrades RNA primers in euks? in proks?
proks: dna pol i
euks: ribonucleases HI and FEN-1
once HI and FEN-1 remove RNA primers, what fills in the gaps?
DNA pols delta and epsilon, with ligase to catalyze phosphodiester bonds between fragments
what happens to histones during euk replication?
they must be removed/dissociated, but are replaced after the replication fork moves through
which end of the chromosome experiences shortening?
5’, primers are removed but you can’t add to the 5’ end
if there is shortening on one side, what happens on the 3’ end? and what do we do about it?
there is an overhang. there are telomeres made of repeats so you’re not losing important info
what is the Hayflick limit?
the number of times a human cell population divides before stopping
what does telomerase do?
it uses an RNA template to lengthen the parent strand
What are the 3 steps in the telomerase process?
- binding of telomerase to 3’ overhang, RNA template hybridizes to DNA and provides template
- elongation, adding bases to parental strand
- translocation and then more elongation
how far does telomerase extend a strand?
50-60 nucleotides
is there still an overhang after telomerase adds nts?
yes
Why is there no chew back in the chromosome after replication?
the ends are protected by a protein complex called shelterin (which is removed during replication and then re-added)
why don’t we want chew back on the chromosomes after replication?
it will shorten the chromosome much faster
when is telomerase fully turned off?
when a cell no longer divides (senescence)
what are the steps of PCR?
- Denature DNA
- Primers hybridize to DNA template
- 20-40 rounds replication
