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a. Because the DNA double helix is very stable, __ and __ are required to separate them in vitro
b. Two proteins that do this in vivo are __ and __, which are needed to open up the double helix to provide a template
- base pairs are locked in place
- high temperatures
- DNA helicase and single-strand DNA-binding proteins
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i. DNA helicases: first isolated as proteins that __; use __ to propel themselves rapidly along a DNA single strand; they __
1. There are helicase that can unwind in t eh __ or __; but, a helicase moving 5’ to 3’ on the lagging strand is more common
- hydrolyze ATP when they are bound to single strands of DNA
- ATP hydrolysis
- pry the helix apart
- 5’à3’ direction or the 3’à 5’ direction
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i. __ bind tightly and cooperatively to exposed single-stranded DNA without __; aid helicases by __, thereby preventing the formation of the __ that readily form in single-strand DNA, which can __
Single-strand DNA-binding (SSB) proteins (aka: helix-destabilizing proteins)
covering the bases
stabilizing the single strand and straightening out the regions of single-stranded DNA on the lagging strand template
short hairpin helices
impede the DNA synthesis catalyzed by DNA polymerase
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a. The tendency to __ allows DNA pol (that just finished making an Okazaki fragment) to be __
i. However, it is more difficult for the polymerase to __.
1. To combat this, there is a __ that keeps polymerase firmly on the DNA when it is moving, but release it as soon as the polymerase runs into a double-stranded region of DNA
- dissociate from a DNA molecule
- recycled
- synthesize the long DNA strands produced at a replication fork and rapidly dissociate
- sliding clamp protein
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a. The Sliding clamp forms a __. One side binds to the __, and the whole ring slides __
i. The assembly of the clamp around the DNA requires __ by a special protein complex, the __, which __ as it loads the clamp on to a __
- ring around the DNA double helix
- back of the DNA polymerase
- freely along the DNA as the polymerase moves
- ATP hydrolysis
- clamp loader
- hydrolyzes ATP
- primer-template junction
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a. On the leading strand, the moving DNA pol is __ and __. The DNA pol on the lagging strand remains attached until __, at which point it dissociates from the template. It then uses a __.
bound to the clamp and remains associated for a while
reaching the 5’ end of the preceding fragment
new clamp
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a. Most proteins involved in replication are part of a large multienzyme complex that rapidly synthesizes DNA. The complex remains __and the DNA is __.
i. __ opens the DNA helix
ii. Two __ work at the fork on each strand
iii. __: provides the RNA proimers
- stationary
- threaded through it
- DNA helicase
- DNA pols
- DNA primase
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a. This complex facilitates the __ each time an __ is synthesized: the __ and the __ are kept in place as a part of the protein machine even when they detach from their DNA template. It also __.
b. __ increase the rate of spontaneous mutation. One such mutant makes a defective form of the__ that is a part of the DNA polymerase enzyme
- loading of the polymerase clamp
- Okazaki fragment
- clamp loader
- lagging strand DNA polymerase molecule
- increae efficiency
- Mutator genes
- 3’-to-5’ proofreading exonuclease
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a. One proofreading system that removes replication errors made by the polymerase that were missed by __ is the __, which detects the potential for distortion in the DNA helix from the __.
i. The system must be able to __, where the replication error occurred.
- proofreading exonucleases
- strand-directed mismatch repair system
- misfit between noncomplementary base pairs
- distinguish and remove the mismatched nucleotide only on the newly synthesized strand
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a. The __ used by the mismatch proofreading system in E. coli depends on __of __ in DNA.
i. __ are added to all A residues in the sequence GATC, but not until after A has been incorporated.
1. As a result, the only GATC sequences that have not been methylated yet are in the __. The recognition of these __ allows the new DNA strands to be transiently distinguished from old ones
- strand-distinction mechanism
- methlation
- selected A residues
- Methyl groups
- new strands just behind a replication fork.
- unmethylated GATCs
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a. The three step process involves __, __, and __.
recognition of a mismatch, excision of the segment of DNA containing the mismatch from the newly synthesized strand, and resynthesis of the excised segment using the old strand as a template.
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a. There is a similar mechanism in umans that helps with predispositions to certain types of cancer, which is due to a __
i. Because most of us __, we are protected
In eucaryotes, to distinguish from the parental strand at the site of a mismatch, __is not needed
defective copy of a mismatch repair gene
inherit two good copies of each gene that encodes a mismatch proofreading protein
methlation
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1. Instead, newly synthesized lagging-strand DNA contains __; and, these __ provide a signal that directs the __
a. This idea requires that the newly synthesized DNA on the leading strand be transiently __.
- nicks (before they are sealed by DNA ligase)
- nicks (single-strand breaks
- mismatch proofreading system to the appropriate strand
- nicked
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a. Because DNA gets tangled during unwinding and too much energy is required to prevent it, a __is formed in the helix by proteins known as __, which adds itself __ to a __, thereby breaking a __. This is reversible and the phosphodiester bond reforms when the protein leaves
- swivel
- DNA topoisomerases
- covalently to a DNA backbone phosphate
- phosphodiester bond in a DNA strand
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i. Topoisomerase I: __
produces a transient single-strand break (or nick), which allows the two sections of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point
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1. Any __in the DNA helix will drive this rotation in the direction that relieves the tension
a. As a result, DNA replication can occur with the __—the part just ahead of the fork
i. Because the __ that joins the __ to a __ retains the energy of the cleaved __, resealing is rapid and does not require additional energy input
ii. In this respect, the rejoining mechanism differs from that catalyzed by the enzyme DNA ligase
- tension
- rotation of only a short length of helix
- covalent linkage
- DNA topoisomerase protein
- DNA phosphate
- phosphodiester bond
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i. Topoisomerase II: __
forms a covalent linkage to both strands of the DNA helix at the same time, making a transient double-strand break in the helix
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1. These enzymes are activated by sites on chromosomes where __
2. Once one binds to such a crossing site, the protein uses __ to perform the following set of reactions efficiently:
a. It __ to create a __
b. It causes the second, nearby double helix to __
c. It then reseals the break and dissociates from the DNA
3. Topoisomerase II can separate __
- two double helices cross over each other
- ATP hydrolysis
- breaks one double helix reversibly
- DNA “gate”
- pass through this break
- two interlocked DNA circles
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