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What are problems in replicating DNA that must be overcome?
- 1) maitain downstream structure
- 2) avoid supercoiling
- 3) antiparallel
- 4) fill in gaps
- 5) H bonds-- melt and reform
- 6) mistakes
- 7) speed
What was done to determine that OF were made on the allging strand?
Use H3-thymidine pulse-chase eperiment
DNA in a cell free system with building blocks, including radioactive thymidine, and all enzymes.
Start the process. Let the replication. Then, add a quick pulse where labeled thymidine is exposed. Let it go for a period of time and then flood the system with cold T to get rid of it called the chase
pulse chase experiemt
done with things we can't see well
begins with no exposure to radioactive label. Then, in a pulse period, it exposes it to the radioactive label
The chase is the period whre there is no more use of hte radioactive adn the nonradioactive is used
Used to determine OF and the replication fork
What are the three requirements for DNA polymerase?
single strand DNA template strand
building blocks: dNTPs
free 3' OH end; DNA pol can't start on own
six subunits: each subunit hydrolyzes ATP
What drives the addition of nucleotides?
the energy form ATP hydrolysis and the triphosphate allows the reaction to proceed
- DNA dependent RNA polymerase
- depends on DNA template to synthesize a complemnetary RNA strand; capable of starting from scratch of a short stretch of RNA
Explain nick sealing.
DNA ligase fixes teh nick. It links together strands of DNA using energy through ATP
DNA ligase finds broken region, grabs ATP, separates phosphates and repairs nick by fusing ATP to 5' end
responsible for the assembly of the clamp around DNA and providing the energy; five subunits; tightens around the primer junction and hydrolyzes ATP and loads the clamp; dissociates onece the clamp has been assembled
keeps polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-stranded region of DNA
What are the sites of DNA Pol?
A polymerizing site and an editing site (exonucleolytic)
In the editing mode, the newly synthesized DNA transiently unpairs from the template and the polymerase undergoes a conformational change, moving the editing catalytic site into place to remove the most recently added nucleotide
What can repair enzymes detect?
they scan along looking for bumps, incorporated as a mistake in the nucleotide sequence
proteins latch on to get rid of strand part containing the bump. A nick is formed adn filled in with nucleotides
Why is it in teh 5-->3 and not 3-->5?
It allows the chain to continue to be elongated when a mistake in polymerizaton has been removed by exonucleolytic proofreading.
In the three to five direction, there would be blockage of further chain elongation. No, high energy bond would be cleaved, as opposed to if it were 5-->3, where a high energy bond can be cleaved
usd to transfer DNA from one organism to another
What was the experiment done to determine origins?
Buffer put over DNA with enzymes to begin the process
After a few minutes, put H3-thymidine and pulse for ten minutes.
Wash away in the chase to reduce the levels of radioactive thymidine
demonstrated that the replication was moving away from the bubble in both directions, tapering off the farther along it went
What is unique about the nucleotide sequnece at the replication origin?
it contains sequence that attracts initiator proteins, which attract helicase in an inactive form (protein bound to it)
An intermediate causes the inhibitor to be released and activated. Primase is then called.
Initiator binds to replication origin. Helicase in inactive form binds to initiator protien. Helicase loaded onto strand and activated, causing opening of the helix and binding of primase to form a primosome. DNA primase lays down primer. DNA pol starts the chain
What was the experiment done with yeast cells?
Colonies of yeast without ability to make histidine loaded on medium lacking histidine.
Genome cut into pieces and introduced into plasmids genetically modified with histdine. Plasmid put in yeast cells so it can make its own histidine in the plasmids are the fragments of the genome.
One fragment will have the origin of replication and cells with plasmid without origin don't pass histidine on
What is inclued in the origin of replicaiton?
there is an ORC binding site, where the origin replication complex will bind
unwinding region, a region containing A-T sequences that can be easily unbound
Abf1-binding site, where an auxilary protein, Abf, facilitates ORC binding (helper proteins help recruit ORC)
ex: NAP-1 and CAF-1 restore the full complement of histones to daughter molecules
RNA-dependent DNA polymerase: template being used is an RNA molecule that is bound to the protein; acts as a base-pairing enzyme
What can occur on a daily basis?
- depurination: N-glycosyl linkages to deoxyribose hydrolyze
- deamination: cytosol converts to uracil
What effect can deamination have?
one daughter strand will inherit a point mutaton
What effect can depurination have?
a deletion can occur in a daughter strand
functions in base excision repair to remove the base using a flipping out method; hydrolyzes the base from the sugar and removes it
along with phosphodiesterase removes the sugar phosphate
Nucleotide excision repairs involves what?
latching on of the enzyme upstream and dounstream and nicks the backbone on either side
a helicase removes the damaged region
larger gap: polymerase latches on to synthesize new strand
What are the two types of homologous recombination?
homologous: most frequently occurs in newly replicated DNA because sister chromatids still associated with each other; damage to one allows the other to fix it; the info exists on sister chromatids; used to fill in gap
non-homologous: chromosome by itself that somehow a ds break has occurred; repair system finds it but results in loss of info; rescuing a damaged chromosome; if nick in both strands, blunt ends are created and joined--> loss of information
Explain non-homologous recombination.
Ku proteins (heterodimers that grasph broken chromosome ends) scan the genome and look for ds breaks.
Binds to the end of the break and recruits additional proteins, which hold the broken ends together while they are processed and joined covalently
The checkpoint can't be passed until all fo the damage is fixed.
Explain homologous recombination.
When a moving replication fork encounters a single-strand break, it will collapse but can be repaired by homologous recombination.
The initial strand invasion requires a free 3' end generated by a nuclease that degrades the 5' end of the complementary strand. Recombination then begins with starnd invasion in which the strand being elongated uses the sister chromatid strand, whih is undamaged. Strand breakage then occurs, along with additional DNA synthesis. The replicaton fork then restarts
How to hybridize strands.
DNA double helices reform from their separated strands in a reaction that depends on the random collision of two complementary DNA strands.
What happens in homologous DNA repair?
RecA holds a double strand and a single strand. First, a non-base-paired complex is formed, which is converted into a joint molecule as a homologous sequence is found.
The RecA positions the strand so that it invades the other.
Can be either spontaneous branch migration or protein-directed branch migratio.
In spontaneous branch migration, there is a random-walk process and makes little progress
Protein directed requires ATP and moves the branch point at a uniform rate in one direction
region where we have crossing over of chromatin; if rotation, you get recombinant DNA
the two DNA strands switch partners between two double helices
cutting of the strands in Holliday junction
Explain homologous recombination in meiosis.
Rather than a randomly occurring break, there is a purposeful ds break.
One chromosome is cut and an exonuclease exposes the single stranded 3' end. Strand invasion joins the chromosomes, creating a double Hilliday junciton.
Two things can occur. The chromosomes can be cut directly, leading to a single region of crossover. Or, they can cut in different places, leading to several crossover regions