Key terms
What related term is sometimes used to refer collectively to many nucleosomes together
Chromatin
What type of supercoiling is more common in vivo?
Negative
Define: Helicase
Enzyme that unzips the double helix at the replication fork
Why is fluorescent DNA sequencing beneficial to use over conventional dideoxy sequencing?
It does not use a radioactive isotope and it can use all 4 ddNTP's in the same reaction.
Define: SSB
- Single stranded binding protein
- prevents strands from snapping back together
- More found on lagging strand (because there is more single stranded DNA)
- Not an enzyme because it doesn't catalyze an actual chemical reaction and because you need more of it with an increasing amount of DNA (an enzyme wouldn't need more)
Define: DNA ligase
Joins DNA fragments together at the end of maturation
What does negative supercoiling have to do with DNA replication?
Behind the replication fork, the daughter double-helices are negatively supercoiled. Pre-existing negative supercoiling makes it easier to open a bubble at an Origin site to initiate replication.
How is the origin site different in prokaryotes vs eukaryotes?
Eukaryotes have several origin sites per chromosome; prokaryotes only have one.
In DNA sequencing, list key differences that pyrosequencing has vs dideoxy sequencing.
Pyrosequencing is a “sequencing by synthesis” method, NOT a chain-termination like dideoxy sequencing
Dideoxy Sequencing has to do 4 different reactions with each ddNTP, while pyrosequencing can read many sequences in parallel
What does ligase do in replication? Why is it needed?
Ligase is an enzyme that reseals nicks in the DNA strands. It is needed so that two separate sections of the DNA strand can join together into one continuous strand.
3'-5' exonuclease
An enzyme that works on nucleic acids one at a time from the 3' end. Functions in proofreading
How are positive and negative supercoiling similar to each other?
Both are going away from a relaxed state. They both cause the formation of superhelical writhes.
What happens at an Origin site?
A pre-replication protein complex recognizes a particular DNA sequence and opens a bubble in the DNA. Helicase and SSB separate and keep apart the strands and extend the bubble. RNA polymerase starts an RNA primer and allows DNA replication to begin.
Compare and contrast dideoxy sequencing and PCR
Compare: dideoxy sequencing and PCR both start with a specific primer
Contrast: PCR does not require ddNTPs while dideoxy sequencing does
DNA sequencing is the process of determining the precise order of the nucleotides in a given DNA fragment while the PCR process creates thousands to millions of copies of the interested DNA fragment.
What strand participates in discontinuous synthesis during replication?
Lagging strand (Okazaki fragments!)
Define: Ring clamp
Clamps the DNA (holds polymerases onto the DNA). Responsible for processivity.
What does positive supercoiling have to do with DNA replication?
It often builds up in front of the replication fork, as helicase crams the same number of twists into a shorter number of base-pairs.
Would you expect an Origin site to be rich in GC base-pairs? Why or why not?
We would expect the origin site to be AT rich (not GC rich) because AT rich regions are easier to pull apart, and the origin needs to be able to pull apart easily so that things like transcription can take place there.
Compare and contrast fluorescent dideoxy sequencing with 32P sequencing.
Compare: Both techniques use dideoxy “terminator” nucleotides and DNA polymerase to generate fragments of varying lengths that precisely identify (by electrophoresis) the last base added.
Contrast:
32P sequencing can read 250-500 bases at a time, while fluorescent dideoxy sequencing can read up to 1000.
32P uses radioactive phosphorus, while fluorescent DNA sequencing uses a fluorescent molecule that is not radioactive.
Instead of using 4 different reactions per piece of sequencing like in 32p, fluorescent sequencing can use all 4 dNTPs in the same reaction.
What is processivity? How do cells ensure a high degree of processivity during replication?
Processivity is the tendency for an enzyme to act repeatedly, without diffusing away and coming back. In DNA replication, a ring clamp encircles each of the DNA polymerases onto the DNA double-helix that it is working on, so that it is literally locked onto its substrate (without being covalently attached).
Define: semi-conservative
each daughter helix gets one old strand and one new strand
What do topoisomerases have to do with DNA replication?
Topoisomerases relax positive supercoiling ahead of the replication fork and relax most of the extreme negative supercoiling present immediately after DNA replication.
What does RNA polymerase do in replication? Why is it needed?
RNA polymerase is an enzyme that synthesizes RNA from DNa template through a process called transcription. It is not only responsible for copying DNA into RNA, but it functions in proofreading. Whenever DNA pol adds a mismatched base by mistake, it pauses and uses its exonuclease tool to remove the mistaken base, then tries again to put in a new (and hopefully correct) base.
What template strand would give rise to the following 32P dideoxy sequencing gel results?
Label the 5’ end of your template strand.
(-) ddA | |
(-) ddT || | | | ||
(-) ddC ||| || | |
(-) ddG || ||
Template: 3'-GGGTAACCAGGCCAGAGAA- 5'
How does proofreading of newly synthesized DNA occur?
Whenever DNA pol adds a mismatched base by mistake, RNA pol pauses and uses its exonuclease tool to remove the mistaken base, then tries again to put in a new (and hopefully correct) base.