Topic 1C Flashcards
what are key elements of info needed to understand DNA replication?
strands are antiparallel, purine base pairs with pyrimidine base
template strand
in DNA replication, the parental strand whose sequence is used to synthesize a complementary daughter strand
daughter strand
in DNA replication, the strand synthesized from a parental template strand
semiconservative replication
the mechanism of DNA replication in which each strand of a parental DNA duplex serves as a template for the synthesis of a new daughter strand
after replication, what does each new DNA duplex consist of?
one strand that was originally part of the parental duplex and one newly synthesized strand
what does conservative replication (the alternative model) propose?
the original DNA duplex remains intact and the daughter DNA duplex is completely new
who’s research and experimental evidence supported semiconservative replication?
Meselson and Stahl-used two non-radioactive isotopes of nitrogen and measured density of DNA
replication fork
the site where the parental DNA strands separate as the DNA duplex unwinds
new DNA strands grows in what direction?
can only grow by adding successive nucleotides to the 3’end-grows in a 5’ to 3’ direction
DNA polymerase
an enzyme that is a critical component of a large protein complex that carries out DNA replication (highly conserved)- synthesizes a new DNA strand from an existing template*
what can correct mistakes in replication?
DNA polymerase
leading strand
a daughter strand that has its 3’ end pointed toward the replication fork, so as the parental double helix unwinds, this daughter strand can be synthesized as one long, continuous polymer
lagging strand
a daughter strand that has its 5’ end pointed toward the replication fork, so as the parental double helix unwinds, a new DNA piece is initiated at intervals, and each new piece is elongated at its 3’ end until it reaches the piece in front of it
Okazaki fragment
in DNA replication, any of the many short DNA pieces in the lagging strand
what explains the presence of leading and lagging strands during DNA replication?
the antiparallel nature of the two strands in a DNA double helix and the fact that DNA polymerase can synthesize DNA in only one direction
primer
a short stretch of RNA at the beginning of each new DNA strand that serves as a starter for DNA synthesis
why is a primer needed?
because DNA polymerase cannot begin a new strand on its own, can only elongate the end of an existing piece of DNA or RNA
RNA primase
an RNA polymerase that synthesizes a short piece of RNA complementary to the DNA template and does not require a primer
where is the section of RNA primer located?
at the 5’ end, the lagging strand has many primers for each fragment
in the lagging strand, when a growing fragment comes into contact with the primer, what happens?
a different DNA polymerase removes RNA primer and extends fragment of DNA to fill space left over. DNA ligase joins fragments
DNA ligase
an enzyme that uses the energy in ATP to close a nick in a DNA strand, joining the 3’ hydroxyl of one end to the ‘ phosphate of the other end
topoisomerase II
an enzyme that breaks a DNA helix, rotates the ends and seals the break-relieves stress on the double helix that results from its unwinding at the replication fork
helicase
a protein that unwinds the parental double helix at the replication fork
single-strand binding protein
a protein that binds single-stranded nucleic acids-prevents template strands from coming back together
how does synthesis of DNA strands occur at the same time and rate?
the synthesis is coordinated because: polymerase complexes for each strand stay in contact with each other-polymerase passes through in the same direction, lagging strand is looped until new legging strand encounters previous fragment: 3’ end of both strands are elongated together
what happens to rate of replication when DNA damage occurs?
rate of synthesis slows down so that DNA can be repaired
if synthesis of one strand slows down to repair DNA damage, what happens to the other strand?
the synthesis slows down too
when is pairing of the replication complexes disrupted?
when the RNA primer of the previous Okazaki fragment is encountered and when new lagging-strand primer is formed
proofreading
the process in which DNA polymerases can immediately correct their own errors by excising and replacing a mismatched base
origin of replication
any point on a DNA molecule at which DNA synthesis is initiated
replication bubble
a region formed by the opening of a DNA duplex at an origin of replication, which has a replication fork at each end
while prokaryotic chromosomes only have …
one origin of replication, eukaryotic chromosomes have many
each replication bubble has:
two replication forks that move in opposite direction
within a single replication bubble, the same daughter strand is:
a leading strand at one replication fork and the lagging strand at the other replication fork
circular DNA molecules typically have:
only one origin of replication
circular DNA molecules can be replicated completely because:
it has no ends, replication forks move completely around the circle
linear DNA molecules have ends, and after each round of DNA replication….
the ends become slightly shorter
where is a primer required in the leading strand?
only at the origin of replication
what is the purpose of the final RNA primer in the lagging strand?
this primer initiates synthesis of the final Okazaki fragment of the lagging strand, however this means that there is no other Okazaki fragment to synthesize the missing 100 base pairs and remove the primer
telomere
a repeating sequence at each end of a eukaryotic chromosome (attaches to template strand-3’ end, allows formation of original telomere sequence in daughter strand)
telomerase
an enzyme containing an RNA template from which complementary telomere repeats are synthesized
once the 3’ end of the template strand has been elongated by the telomere, …
an RNA primer and the complementary DNA strands are synthesized to restore the original telomere which can synthesize another length of the original telomere in daughter strand
the slight shortening and subsequent restoration that takes place has no harmful consequences because…
telomeres have no genes
germ cells
the reproductive cells that produce sperm or eggs and the cells that give rise to them
stem cell
an undifferentiated cell that can undergo an unlimited number of mitotic divisions and differentiate into any of a large number of specialized cell types
where is telomerase activity most active?
in germ cells and stem cells
describe telomerase activity in most cells in the adult body:
inactive-telomere is actually shortened 100 base pairs in each mitotic division which limits the number of mitotic divisions that cells can undergo
human cells stop dividing when:
their chromosomes have telomeres with fewer than about 100 copies of the telomere repeat
what is the telomere repeat sequence?
3’-GGGATT-5’
polymerase chain reaction (PCR)
a selective and highly sensitive method for making copies of a piece of DNA, which allows a targeted region of a DNA molecule to be replicated into as many copies as desired
amplified
in PCR technology, an alternative term for replicated
when is PCR used? (hint: PCR is both selective and highly sensitive)
it can be used to amplify and detect small quantities of nucleic acids
what does the PCR solution require:
template DNA (including region to be amplified), DNA polymerase, all nucleotides, primers (amount greater than number of template DNA molecules)
oligonucleotide
a short (typically 20 to 30 nucleotides), single-stranded molecule of known sequence produced by chemical synthesis; oligonucleotides are often used as primer sequences in the polymerase chain reaction
the base sequences of oligonucleotides are chosen to be:
complementary to the ends of the region of DNA to be amplified
what are the three steps of PCR?
- denaturation
- annealing
- extension
denaturation
the unfolding of proteins by chemical treatment or high temperature; the separation of paired, complementary strands of nucleic acid (breaks H-bonds*)
annealing
the coming together of complementary strands of single-stranded nucleic acids by base pairing-primers anneal to complementary sequence on the DNA
extension (PCR)
a step in the PCR for producing new DNA fragments in which the reaction mixture is heated to the optimal temperature for DNA polymerase, and each primer is elongated by means of deoxynucleoside triphosphates
PCR is repeated over and over (25-35 cycles) and the number of copies of the targeted fragment…
doubles (2^n), after third round of amplification, only molecules as long as the region of the template that is complementary to the primers is produced
why do you have to continue adding fresh DNA polymerase?
because the enzyme loses structure and function at the high temperatures during the denaturation stage
in modern times, DNA polymerase from what bacteria is used?
Taz polymerase (resistant to heat, lived in hot springs)
how do you determine whether or not PCR has yielded the expected product?
by determining the size of the amplified DNA molecules (compares expected size to actual size-by gel electrophoresis)
gel electrophoresis
a procedure to determine the size of a DNA fragment, in which DNA samples are inserted into slots or wells in a gel and a current is passed through. Fragments move toward the positive pole according to size
in a sample of gel electrophoresis, where are the larger fragments located?
near the top, moves less in a given interval of time compared to shorter fragments
