Week 4 Textbook Reading Flashcards
what would happen if incorrect base pairs are allowed to remain?
Incorrect base pairs are formed much less frequently than correct ones, but if allowed to remain, they would result in an accumulation of mutations
what 2 special qualities does DNA polymerase have that increases the accuracy of DNA replication
- The enzyme carefully monitors the base-pairing between each incoming nucleoside triphosphate and the template strand
Only when the match is correct does DNA polymerase undergo a small structural rearrangement that allows it to re-catalyze the nucleotide-addition rxn - When DNA polymerase does make a mistake and adds the wrong nucleotide, it can correct the error through an activity called proofreading
proofreading
Proofreading takes place at the same time as DNA synthesis
-Before the enzyme adds the next nucleotide to a growing DNA strand, it checks whether the previously added nucleotide is correctly base-paired to the template strand
–If so, the polymerase adds the next nucleotide
–If not, the polymerase pauses to clip off the mispaired nucleotide and then tries again
who does the proofreading performed in dna
dna polymerases
If a DNA polymerase were to synthesize in the reverse direction, what would happen?
If a DNA polymerase were to synthesize in the reverse direction, it would be unable to proofread
-This is because if this “backward” polymerase were to remove an incorrectly paired nucleotide from the 5’ end, it would create a chemical dead end- a strand that can no longer be elongated
How can polymerase begin a completely new DNA strand?
To get the process started, a different enzyme called primase begins a new polynucleotide strand by joining two nucleotides together without the need for a base-paired end
-RNA polymerase that uses DNA as a template to produce a short RNA fragment that serves as a primer for DNA synthesis
–Primase is an example of an RNA polymerase, an enzyme that synthesizes RNA using DNA as a template
what is needed on the leading strand
For the leading strand, an RNA primer is needed only to start replication at a replication origin
At that point, the DNA polymerase simply takes over, extending this primer with DNA synthesized in the 5’ to 3’ direction
what is needed to produce okazaki fragments?
DNA polymerase then adds a deoxyribonucleotide to the 3’ end of each new primer to produce another fragment
It will continue to elongate this fragment until it runs into the previously synthesized RNA primer
To produce a continuous new DNA strand from the pieces on the lagging strand:
3 additional enzymes are needed
A nuclease degrades the RNA primer
DNA polymerase called a repair polymerase replaces the RNA primers with DNA (using the end of the Okazaki fragment as its primer)
DNA ligase joins the 5’ phosphate end of one DNA fragment to the adjacent 3’-hydroxyl end of the next
The repair polymerase involved in this process is DNA polymerase I
nuclease
degrades the RNA primer
dna polymerase
DNA polymerase called a repair polymerase replaces the RNA primers with DNA (using the end of the Okazaki fragment as its primer)
DNA ligase
DNA ligase joins the 5’ phosphate end of one DNA fragment to the adjacent 3’-hydroxyl end of the next
DNA polymerase iii
The polymerase that carries out the bulk of DNA replication at the forks is known as DNA polymerase iii
why does primase often make mistakes and how are they detected?
Unlike DNA polymerases i and iii, primase does not proofread its work and can often make mistakes
Since they’re made out of RNA, their mistakes are easily noticeable and can be easily be removed
for dna rep. to occur, the double helix must…
For DNA replication to occur, the double helix must be continuously pried apart so that the incoming nucleoside triphosphates can form base pairs with each template strand
2 types of replication proteins- DNA helicases and single-strand DNA-binding proteins- cooperate to carry out this task
dna helicase
DNA helicase is an enzyme that pries open the DNA double helix, using energy derived from ATP hydrolysis
Used to expose DNA single strands for DNA replication
single strand binding proteins
Single strand binding proteins then latch onto the single-stranded DNA exposed by the helicase, preventing the strands from re-forming base pairs and keeping them in an elongated form so that they can serve as templates
what problem does the unwinding of the dna double helix create?
As the helicase moves forward, prying open the double helix, the DNA ahead of the fork gets wound more tightly
This excess twisting in front of the replication fork creates a tension in the DNA that- if allowed to build- would make unwinding the double helix difficult and impede the forward movement of the replication machinery
Enzymes called DNA topoisomerases relieve this tension and produces a single-strand break in the DNA backbone, which releases the built-up tension
dna topoisomerases
Enzymes called DNA topoisomerases relieve this tension and produces a single-strand break in the DNA backbone, which releases the built-up tension
The enzyme then reseals the nick before falling off the DNA
function of the sliding clamp
It’s a protein that keeps DNA polymerase firmly attached to the template while it is synthesizing new strands of DNA
Left on their own, most DNA polymerase molecules will synthesize only a short string of nucleotides before falling off the DNA template strand
The sliding clamp forms a ring around the newly formed DNA double helix and, by tightly gripping the polymerase, allows the enzyme to move along the template strand without falling off as it synthesizes new DNA
function of the clamp loader
Assembly of the clamp around DNA requires the activity of another replication protein, the clamp loader, which hydrolyzes ATP each time it locks a sliding clamp around a newly formed DNA double helix
This loading needs to happen once per replication cycle on the leading strand
On the lagging strand, the clamp is removed and reattached each time a new Okazaki fragment is made
telomerase
Because DNA replication proceeds only in the 5 to 3 direction, the lagging strand of the replication fork must be synthesized in the form of discontinuous DNA fragments, each of which is initiated from an RNA primer laid down by a primase
What problem appears as the replication fork approaches the end of a chromosome?
Although the leading strand can be replicated all the way down to the chromosome tip, the lagging strand cannot
When the final RNA primer on the lagging strand is removed, there is no enzyme that can replace it with DNA
Eukaryotes get around this by adding long, repetitive nucleotide sequences to the ends of every chromosome, providing the replication machinery with “extra” DNA to complete the lagging strand
These sequences, which are incorporated into structures called telomeres, attract an enzyme called telomerase to the chromosome ends
telomeres and telomerase
These sequences, which are incorporated into structures called telomeres, attract an enzyme called telomerase to the chromosome ends
Telomerase carries its own RNA template, which it uses to add multiple copies of the same repetitive DNA sequence to the lagging-strand template
In many dividing cells, telomeres are continuously replenished and the resulting extended templates can then be copied by DNA replication, ensuring that no peripheral chromosomal sequences are lost
what causes cells to stop dividing
After many rounds of cell division, the telomeres in descendant cells will shrink, until they disappear
At this point, the cells will cease dividing
dna repair
Most DNA damage is only temporary, because it is immediately corrected by processes collectively called DNA repair
depurination
Depurination is a spontaneous rxn that doesn’t break the DNA phosphodiester backbone but removes a purine base from a nucleotide
Gives rise to lesions that resemble missing teeth
deamination
Deamination is a rxn with water that causes the spontaneous loss of an amino group from a cytosine in DNA to produce the base uracil
how is uv radiation in sunlight damaging to dna
It promotes covalent linkage between 2 adjacent pyrimidine bases, forming the thymine dimer
basic steps in dna repair process
Top strand is damaged → segment of damaged strand is excised→repair DNA polymerase fills in missing nucleotide in top strand using bottom strand as template→DNA ligase seals nick→DNA damage repaired
detailed steps in dna repair process
The damaged DNA is recognized and removed by nucleases that cut the DNA backbone around the damage, leaving a small gap on one strand of the double helix
A repair DNA polymerase binds to the 3’-hydroxyl end of the cut DNA strand
-The enzyme then fills in the gap by making complementary copy of the info present in the undamaged strand
-Repair DNA polymerases synthesize DNA strands by elongating chains and proofreading
-Repair polymerase then fills in gaps left after the RNA primers are removed during the normal DNA replication process
When the repair DNA polymerase has filled in the gap, a break remains in the sugar-phosphate backbone of the repaired strand
-This break is sealed by DNA ligase, the same enzyme that joins the Okazaki fragments during replication of the lagging DNA strand
mismatch repair
A DNA mismatch repair system removes replication errors that escape proofreading
Mismatch repair is a mechanism for recognizing and correcting incorrectly paired nucleotides- those that are non complementary
what happens when the replication machinery makes a copying mistake..
Whenever the replication machinery makes a copying mistake, it leaves behind a mispaired nucleotide
-If left uncorrected, the mismatch will result in a permanent mutation in the next round of DNA replication
-In most cases, a complex of mismatch repair proteins will detect the DNA mismatch, remove a portion of the newly synthesized DNA strand containing the error and then resynthesize the missing DNA
–This repair mechanism restores the correct sequence
To be effective, the mismatch repair system must be able to recognize …
To be effective, the mismatch repair system must be able to recognize which of the 2 DNA strands contains the error
-Removing a segment from the strand that contains the correct sequence would only compound the mistake
–The mismatch system solves this problem by recognizing and removing only the newly made DNA
—In bacteria, newly synthesized DNA lacks a type of chemical modification (a methyl group added to certain adenines) that’s present on the preexisting parent DNA
—-Newly synthesized DNA is unmethylated for a short time, during which the new and template strands can be easily distinguished
what role does mismatch repair play in preventing cancer in humans
Some cancers are caused by mutations in genes that encode mismatch repair proteins
Individuals who inherit a single damaged mismatch repair gene are unaffected until the undamaged copy of the same gene is randomly mutated in a somatic cell
This mutant cell- and all of its progeny- are then deficient in mismatch repair
They therefore accumulate mutations more rapidly than do normal cells
Because cancers arise from cells that have accumulated multiple mutations, a cell deficient in mismatch repair has a greatly enhanced change of becoming cancerous
What happens when both strands of a DNA segment are damaged at the same time?
Mishaps at the replication fork, radiation, and various chemical assaults can all fracture DNA, creating a double-strand break
These are dangerous because they can lead to fragmentation of chromosomes and the subsequent loss of genes
Difficult to repair
If a chromosome experiences this break, and the broken pieces become separated, the cell has no nearby copy it can use as a template to reconstruct the lost info
how do cells repair double-strand breaks
To handle this type of DNA damage, cells have evolved 2 basic strategies:
The first involves sticking the broken ends back together, before the DNA fragments drift apart and get lost
-This repair mechanism, called nonhomologous end joining, occurs in many cell types and is carried out by a specialized group of enzymes that “clean up” the broken ends and rejoin them by DNA ligation
–The cost of this is, in “cleaning up” the break to make it ready for ligation, nucleotides are often lost at the site of repair
Fortunately, cells have an alternative, error-free strategy for repairing double-strand breaks, called homologous recombination
what can result due to failure to repair dna damage
Failure to repair DNA damage can have severe consequences for a cell or organism
If an error occurs in a particular position in the DNA sequence, it could alter the AA sequence of a protein in a way that reduces or eliminates that protein’s ability to function
The example of sickle-cell anemia, which’s an inherited disease, illustrates the consequence of mutations arising in the reproductive germ-line cells
A mutation in a germ-line cell will be passed on to all the cells in the body of the multi-cellular organism that develop from it, including the gametes responsible for the production of the next gen
In extreme cases, an unchecked cell proliferation results in…
In extreme cases, an unchecked cell proliferation known as cancer results
-Caused by an accumulation of random mutations in a somatic cell and its descendants
–Increasing the mutation frequency even 2 or 3 fold could cause a disastrous increase by accelerating the rate at which such somatic cell variants arise
