Theme 1C Flashcards
are prokaryotic DNA molecules single stranded
no, they have one, circular, double-stranded DNA molecule
what is the origin of replication (ori)
the starting site of DNA replication, multiple log DNA molecule
what is the replication bubble
the space that forms as the parental strands separate
what are replication forks
the edges of a replication bubble, each bubble has two
where does DNA polymerization happen
the replication forks
what does the enzyme helicase do
it untwists the double helix at each replication fork (separates the template strands)
what does the single-stranded binding protein (SSB) do
it binds the ssDNA and prevents them from re-annealing before replication
what does topoisomerase do
it releases the tension(supercoiling) caused by helicase
where can DNA polymerase add new nucelotides
a 3’ -OH of a double stranded molecule
what does primase do
it synthesizes a short RNA primer that can add RNA nucleotides without a 3’-OH
after primase adds 10-20 RNA nucleotides paired with the template strand, what is left
a DNA/RNA hybrid
what does DNA polymerase III do
adds ddNTPs (deoxynucleotide triphosphates), starting at the 3’-OH end of the RNA primer
which direction is the template strand read by DNA polymerase III
3’-5’
what direction does DNA polymerase III add dNTPs to the new strand
5’ - 3’
what does the DNA sliding clamp do
it stabilizes the DNA polymerase so it does not fall off the template strand
which direction is the lagging strand replicated in (relative to the replication fork)
away from the replication fork (fork must advance before replication can happen
what are the short segments synthesized in the replication of the lagging strand called
Okazaki fragments
after the replication bubbles merge, what is left
2 double stranded DNA molecules, each containing one parental strand and one daughter strand (that still has the RNA primers)
what does DNA polymerase I do
removes the RNA nucleotides and replaces them with DNA molecules
what does DNA ligase do
seals the ‘nicks’ between the last dNTP added by DNA polymerase I and the first dNTP added by DNA polymerase III by forming a phosphodiester bond between them
all the enzymes involved in DNA replication are organized together into a _____________ to increase efficiency of replication
replisome
what were the potential models for DNA replication
semi conservative, conservative, or dispersion
what was used to track parental and newly synthesized DNA strands over several generations
nitrogen isotopes
what was the first part of the experiment that proved the semiconservative model
E.coli was grown with only 15N present (an isotope, heavier than normal) (100% 15N in its nitrogenous bases), the DNA was consolidated and centrifuged, it settled based on its density
what was the second part of the experiment that proved the semiconservative model
E.coli was transferred to a medium with only 14N. all new nucleotides would have 100% 14N, all old nucleotides would have 100% 15N, they sampled the DNA after one replication, then after another
what were the results of the experiment that proved the semiconservative model of DNA replication
after one replication in 14N, they found a 15N-14N hybrid DNA which was lighter than the 15N DNA, after the second replication, there was 50% hybrid DNA and 50% just 14N DNA (the lightest)
what provides energy for the formation of the new phosphodiester bond when nucleotides are being added
hydrolysis of pyrophosphates
what is the end replication problem
the gap left by the RNA primer on the 5’- end of the lagging strand cannot be filled by DNA polymerase because it can only attatch to the 3’-OH. this means after each replication, the DNA strands get shorter
how are the negative effects of the end replication problem mitigated
telomeres (~10,000 bp of non coding DNA sequences) are are both ends of linear chromosomes. ~ 60 are lost each replication
what happenes when there is no more telomere
the cell stops dividing (senescent)
what is telomerase
an enzyme that restores shortened telomeres
where is telomerase found
not in most eukaryotic cells, is present in gamates and stem cells
what are human telomerase mutations used for
as a bio marker in cancers, many cancers acquire mutations that activate telomerase to negate the limitations of rapid cell division
do prokaryotes have more than one origin of replication
no, only one in their circular genome
what is the terminus
where the two sides of the replication bubble meet in a circular DNA molecule
how are plasmids copied
rolling circle replication, one strand is cut by nuclease and the plasmid “rolls” as it replicates both strands, one “unrolling” along the way
what is the lytic cycle
a way of bacteriophage replication, the viral genome replicated using host cell macromolecules and machinery
what is the lysogenic cycle
a way of bacteriophage replication, viral DNA (prophage) inserted in to host cell (lysogen) chromosome, as it replicates, a population of lysogen is formed, eventually prophage comes out and swtiches to the lytic cycle
why is it important to have high fidelity DNA replication
if not, it can cause a defective genome, possibly resulting in disease (like cancer) and death of the organism
what are the three main DNA repair mechanisms
proofreading, mismatch repair, and excision repair
what proofreads and detects mistakes in DNA
DNA polymerase III
how does DNA polymerase III fix mistakes in DNA
it uses its 3’ - 5’ exonuclease activity to remove the most reacently added nucleotides (the mismatched one) and replaces the correct nucleotide, resuming synthesis
what do proofreading exolucleases reduce the ratio of mismatches to
1 in 10 million
what does DNA mismatch repair (MMR) do
fixed replication errors not corrected by proofreading
what recognizes the mismatch in MMR
MutS and MutL (DNA binding proteins)
what does MutH do in MMR
nicks daughter strand several nucleotides away from the mismatch (creates a gap)
what excises the region of daughter strand surrounding the mismatch
exo 1 5’ - 3’ exonuclease
what do DNA plolymerase III and DNA ligase do in MMR
DNA polymerase fills the gap made by exo1 5’-3’ exonuclease an repairs the mismatch, DNA ligase seals the nick closed
what is base-excision repair for
to correct damage made to nitrogenous bases that are damaged by chemical modifications like oxidation, alkylation, or deamination
what removes the modified base, forming and AP site in base excision repair
DNA glycosylases
what does AP endonuclease do in base excision repair
removes the AP site, polymerase repairs the damage
what is nucleotide excision repair
a way to correct damage to the backbone made from exposure to UV radiation or chemical mutagens
what is genomics
the study of the whole genome
determining the base to base sequence of nucleotides
sequencing
determining coding/non-coding parts of a genome
Annotations
determining the function of the coding parts of a genome
Functional analysis
comparing genomes to determine the relationships among organisms
evolutionary analysis
what are the 5 overall steps to sanger sequencing
- DNA purification
- DNA fragmentation
- amplification
- sequencing
- assembly
what is the first step of sanger sequencing
DNA purification, getting rid of other cell macromolecules
what is the second step of sanger sequencing
DAN fragmentation, using restriction enzymes to chop the DNA into smaller pieces
what is the third step of sanger sequencing
amplification, using PCR to make hundreds of copies of each fragment
what is the fourth step of sanger sequencing
sequencing, determining the base sequence of each fragment
what is the fifth step of sanger sequencing
assembly, putting fragments back together
what are dideoxynucleotides
nucleotides that lack a 3’ -OH
what happens when a ddNTP base pairs with a template
DNA polymerase adds to to the daughter strand, but replication stops because additional dNTPs can’t be added without a 3’ -OH
what is Gel Electrophoreisis used for
a way of sequencing genes, used in original sanger sequencing
explain the process of gel electrophoresis
DNA loaded in to a well at one end of a porous matrix (gel). and electric field is generated (- at the top, + at the bottom), DNA migrates to the +, smaller particles move faster
in original sanger sequencing, what happens after electrophoresis
the gel is exposed to x-rays to see the DNA, each lane represents a different nucleotide
how is modern sanger sequencing different from original sanger sequencing
ddNTPs now have fluorescent tags, each of a different wavelength. sequencing can be done in a single test tube instead of 4. it is sent through gel electrophoresis, and the sequence can be determined from the fluorescent patterns
what is PCR
polymerase chain reaction, it is used for DNA amplification
what are the requirements for PCR
- template DNA
- DNA primers (for each strand)
- dNTPs
- heat tolerant DNA polymerase
- PCR machine
what is the process of polymerase chain reaction
- heat sample to 95C (DNA strands separate)
- cool sample to 55C (DNA primers anneal)
- heat sample to 72C (taq polymerase synthesizes daughter strands)
- repeat
