DNA Replication Flashcards
PCR (in vitro) vs. DNA Replication (in vivo)
replicates only the stretch of DNA within the synthesized primers vs. replicates the entire genome
DNA primers synthesized in lab vs. RNA primers built by primase
denature DNA by heating up the mixture vs. helicase and single-stranded binding proteins unwind the DNA
heat tolerant DNA polymerase vs. multiple different DNA polymerases with different jobs aided by a sliding clamp proteins
origins of replication
eukaryotes have multiple ori due to their size
prokaryotes have a single ori since their DNA is stored as plasmids so the ori will reach the whole genome
pre-replication complex binds, initiation begins
replication forks
replication process in both directions from the ori to form replication forks
move away from each other during elongation
leading and lagging strand switch at the ori
helicase and single-stranded binding proteins
involved in initiation
unwinds DNA by breaking hydrogen bonds
the SSBP hold the replication bubble open by tightly binding to the single stranded DNA
primase
adds new RNA bases complimentary to the template strand in order to create a free 3’ end for DNA polymerase III to add bases to
processive
a single enzyme can catalyze the same reaction many times
greater processivity = more times an enzyme can catalyze a reaction before it becomes inactive
DNA Polymerase I, III, RNA polymerase, and primase
sliding DNA clamp
binds to DNA to secure the parent and replicated strand, DNA polymerase binds to the clamp-DNA complex
w/o clamp: dozens of bases
w/ clamp: hundreds of bases
DNA polymerase III
marks the beginning of the elongation period
adds most of the new base to the new strand
continues from the 3’ end of an RNA primer
DNA Polymerase I
- hydrolyzes the RNA primer and replaces it with DNA nucleotides
- ligase connects the gap that exists between the newly added DNA and the previously existing DNA by catalyzing the formation of the phosphodiester bond
- exonuclease and DNA binding ability
Okazaki fragments
DNA fragments generated along the lagging strand
DNA replication can only take place in 5’->3’ direction, when fork opens the opposite way the lagging strand is created
DNA polymerase I replaces multiple primers with DNA and ligase joins the free ends together
telomeres
- highly repetitive noncoding sequences at the end of a chromosome
- prevent chromosomes from being mistakenly joined together by binding to protective proteins and form secondary structures
end replication problem
the lagging strand cannot be fully replicated since there is no space for the DNA polymerase to attach and synthesize DNA
there is no free 3’-OH end to for DNA polymerase I to replace the primer with
stem cells
express the telomerase gene
ribonucleoprotein: complex of RNA and proteins to catalyze reactions and pair bases together
telomerase
uses an RNA template made by primase to extend the telomere (on the DNA template) through base pairing
DNA polymerase III fills the gap between the primer and the existing DNA, DNA polymerase I replaces RNA, ligase closes the DNA gap
Why does the need for RNA primers limit the number of times DNA can be replicated? How does telomerase prevent this?
without the RNA primer, DNA polymerase cannot add nucleotides, so there is an overhang of DNA
DNA must be shortened by cutting the telomere, cell will undergo death when the telomere is completely cut
telomerase prevents this by extending the telomerase
mutations during the cell cycle
errors in replication by DNA polymerase: repaired by proofreading, sometimes permanent
imperfect meiosis: nondisjunction (failure of chromosomes to separate properly) and random breaking and rejoining of chromosomes
proofreading
DNA polymerase III proofreads as it adds bases during replication
proteins of the replication excise the incorrect nucleotide
DNA polymerase III adds the correct nucleotide
Why is the ability to proofread beneficial?
proofreading prevents disease and mutation which allows an organism to live and produce offspring
proofreading ability passed down to offspring which allows them to survive longer
mismatch repair
occurs immediately after DNA replication
DNA polymerase I excises incorrect nucleotide and some adjacent ones and replaces it
ligase connects the gap
excision repair
occurs anytime a mutagen changes a nucleotide
DNA polymerase I excises damaged nucleotide and some adjacent ones
ligase connects the gap
