Chpt 5: DNA replication Flashcards
what is a mutation rate
the rate at which changes occur in DNA sequences
are the amount of damaged genes a good indicator of the amount of mutations
- no
- many mutations are silent
why are the number of essential genes limited
if you have too many, the chances of there being a mutation in an essential gene is catastrophically high
what are the 2 types of cells in a sexually reproducing plant/ animal
- germ transmits genetic info from parent to kid
- somatic forms the body of the organism
what is DNA templating
the mechanism the cell uses to copy the nucleotide sequence of one DNA strand into a complementary DNA sequence
what is the fundamental reaction by which DNA is synthesized
the addition of a deoxyribonucleoside triphosphate to the 3’ end of a polynucleotide chain
is the DNA replication fork symmetrical
no
what direction is DNA synthesized in
5’ to 3’
what forms of proofreading are available
- DNA polymerase
- exonucleolytic proofreading
- strand-direted mismatch repair
describe how DNA polymerase proofreading works
- after complementary nucleotide binding, but before covalently bonding it
- polymerase must go under a conformational change where it tightens around the active site
- this is harder to do when the wrong bases are paired together
- therefore more likely to diffuse away before the polymerase can mistakenly add them
why does DNA replication occur only in the 5’ to 3’ direction
the need for accuracy (if it went from 3’ to 5’, the 5’ side would need to have the energy-full bond to supply the reaction w energy, buuut then any mistakes couldn’t be hydrolyzed away, cause the bare 5’ end would immediately stop the DNA synthesis)
where does the energy for the polymerization reaction that occurs during DNA replication come from
the hydrolysis of high-energy phosphate bond in the incoming nucleoside triphosphate and the release of pyrophosphate
how does DNA polymerase catalyse the reaction that occurs during DNA replication
it positions the incoming nuceloside triphosphate to the template strand
does the leading or lagging strand have more primers
lagging
what makes the RNA primers
DNA primase
describe the process by which the lagging strand works
- RNA primer is synthesized by primase
- DNA polymerase adds nucleotides to 3’ end of RNA primer
- DNA polymerase finishes okazaki fragment
- previous RNA primer removed by nucleases and replaced w DNA by repair DNA polymerase
- nick sealed by DNA ligase
which proteins are needed to open the double helix and present an appropriate single-stranded DNA template for polymerase to copy
- DNA helicases
- DNA-binding proteins
what do DNA helicases do and how
- prys apart the DNA strands
- by hydrolyzing ATP, which then changes the shape of a protein
what do single-strand DNA binding proteins do and how
- straightens out the regions of single stranded DNA
- binds tightly and cooperatively (idk thats all it says)
what holds moving DNA polymerase onto the DNA
- a sliding ring
- called PCNA
- keeps polymerase on DNA, until it reaches a double-stranded portion
describe how DNA polymerase attaches to DNA
- a clamp loader binds to a sliding clamp using ATP
- this opens up the ring shape of the sliding clamp
- the open clamp moves onto DNA
- ATP hydrolysis locks sliding clamp around DNA and releases the clamp loader
- DNA polymerase binds to the sliding clamp
describe the difference in the clamp loader on leading vs lagging strand
- leading clamp and polymerase remain attached for a long time
- lagging they dissociate every time the polymerase reaches the 5’ end of the preceding Okazaki fragment. it will then associate w a new clamp at the next primer
are all the proteins indepedent
nope, most of the time theyre acc in a big complex together
is DNA replication similar in eukaryotes and bacteria
fundamentally yes
how is DNA replication different in bacteria and eukaryotes
- eukaryotes have 3 kinds of DNA polymerase used at the replication fork
- diff detailed structures of their individual protein components (almost completely diff AA sequences)
what are the different polymerases in DNA and how do they differ
- Polε: synthesizes the leading strand, can bind to both sliding clamp and replicative helicate (allowing it to synthesize long stretches of DNA without dissociating)
- Polα and Polδ: synthesize the lagging strand, Polα includes DNA primase as one of its subunits, which is needed more w the lagging strand but can’t add as much bases before dissociating…. Polδ with a sliding clamp can add more nucleotides to finish off the job
what do mutator genes do
greatly increase the rate of spontaneous mutations
describe how strand-directed mismatch repair in eukaryotes work
- MutS protein recognizes and locks onto DNA mismatch
- MutS recruits MutL and scans DNA
- sliding clamp is rencountered, MutL nuclease is activated and initiates strand removal
- strand is removed, and repair DNA synthesis by polymerase δ
what happens if RNA is encorporated into DNA
- it weakens the DNA chain at that point, rendering it highly susceptible to breakage
- this can lead to high mutation rates and genome rearrangements if left unrepaired
what prevents DNA tangling during replication
DNA topoisomerases
how does DNA topoisomerase I work
- produces a transient single-strand break
- this break in the phosphodiester backbone allows the 2 sections of DNA helix on either end to rotate freely
how does DNA topoisomerase II work
- forms a covalent linkage to both strands of the DNA helix at the same time (a transient double-strand break)
- breaks one double helix reversibly to create a DNA “gate”
- causes the second, nearby double ehlix to pass through this opening
- it then reseals the break and dissociates from the DNA
what is the shape of a replication fork
Y
the DNA replication process is started with which special proteins
special initiator ptoreins
the positions at which the DNA helix is first opened are called what
replication origins
roughly how long are replication origins
several hundred nucleotide pairs long
do bacterial chromosomes have one or more replication origins
one
when can e coli control DNA replication
only at initiation (so this is highly regulated)
what is the other name for DNA topoisomerase II
DNA gyrase
how many DNA polymerases are in a bacterial replication fork
2: one on leading and one on lagging
what are 3 bacterial replication proteins
- initiator protein: DnaA
- helicase: DnaB
- primase: DnaG
how does exonucleolytic proofreading by DNA polymerase work
- When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base
- then dna polymerase can keep doing its thing
what does DnaA do
binds to specific sequences at oriC
what does DnaB do
- 2 helicases brought in by helicase loading proteins (DnaC proteins)
- inhibit the helicases until they are properly loaded at oriC
what does DnaG do
synthesizes initial primers
when does DNA replication take place in eukaryotes
- only at one part of the cell cycle
- must be coordinated w mitosis and the cell cyle
does eukaryotic DNA shorten after each replication and why
- yes
- cause when the final primer is removed, there is a section of template that remains unreplicated
- but this is okay cause telomeres exist
what replicates the ends of chromosomes
telomerase
does bacteria DNA shorten after each replication and why
- no
- cause its a circle so there is no “end” to the dna
what is the 5’ end
what is the 3’ end
what does telomerase do
replicates the ends of chromosomes
describe how telomerase replicates the ends of chromosomes
- telomerase binds
- using its RNA component as the template, it adds 2 more repeats to the 3’ end of DNA
- this is done via reverse transcription by telomerase to produce DNA from RNA template
- DNA polymerase can then complimentary bp to it
what is the point of a t-loop
to provide additional protection for the ends of chromosomes
how do t-loops form
by inserting the ends of the chromosome, which is usually 3’ overhang back into the DNA of the chromosome
