DNA REPLICATION Flashcards
What is semiconservative replication of DNA?
the 2 original strands of DNA separate and each acts as a template on which a new complementary strand is laid down
(storage and transmission of genetic information)
Who proved DNA replication is semiconservative?
Meselson and Stahl (1957)
used ecoli and heavy nitrogen
What did Kornberg do in 1958?
descried the purification of an enzyme called DNA polymerase I from Ecoli that synthesized DNA
How is DNA synthesized?
from nucleotide triphosphate precursors by the enzyme DNA polymerase
What did John Cairns in 1963 reveal?
that an ecoli chromosome is a single 1.7mm circular molecule (replicating DNA looks like theta)
How is DNA replicated in bacteria? (remember circular structure)
bidirectionally because a replication fork is found on each end of the loop where DNA is unwound and the daughter strand is being synthesized
(don’t worry too much about this)
What is a primer?
a segment of nucleic acid (RNA or DNA) base paired to the template and with a free 3’-OH
Who discovered the DNA had a template strand and base pairing rules?
Watson-Crick
What is processivity?
number of bases synthesized before the DNA polymerase enzyme dissociates from the template (can be thousands)
What does DNA polymerase exhibit?
processivity
Is DNA polymerase and DNA replication accurate?
yes very accurate
Is DNA replication more accurate in humans or bacteria?
bacteria
What improves accuracy of DNA polymerase and DNA replication?
mismatch repair
What causes tautomers to form?
chemical structure is not absolutely stable, proton rearrangement leads to different chemical forms
What are tautomers?
the same atomic composition with a slightly different chemical structure (changes the pairing characteristics of specific bases)
At any given time roughly how many tautomers are there?
1 in 10,000 molecules are in rare tautomeric form
A cytosine tautomer base pairs accurately with what?
adenine (instead of guanine like it would if it was not a tautomer)
What is the proofreading activity of DNA polymerase I?
3’ to 5’ exonuclease activity
acts ONLY on the base just incorporated (no others)
Does polymerization activity stop when there is an error found?
yes because it has to stop and fix the problem (remove mispaired nucleotide) then it will resume
How many E coli DNA polymerases are there and what are they?
3
DNA polymerase I
DNA polymerase II
DNA polymerase III
What is the function of DNA polymerase I in Ecoli?
mostly for repair
What is the function of DNA polymerase II in Ecoli?
(specialized repair function) don’t need to know
What is the function of DNA polymerase III in Ecoli?
mismatch repair (main DNA replication enzyme)
Which DNA polymerase in Ecoli is very processive?
DNA polymerase III
once it clamps on the DNA it stays on for a long time and adds over half a million bases before it is removed
How many subunits does DNA polymerase III have?
10
What are the 3 genes that encode the 3 DNA polymerases?
polA (correlates with DNA polymerase I), polB (correlates with DNA polymerase II), polC(correlates with DNA polymerase III)
What is the activity of polA and what exactly does it do?
has a 5’ to 3’ exonuclease activity used to get rid of primer RNA during DNA replication
Is polA, polB, or polC the large gene that makes a big donut shaped clamp?
polC
What are the 3 stages of DNA replication?
initiation
elongation
termination
Which stage in DNA replication most complicated?
initiation
Where does DNA replication begin?
origin of replication (ORI)
How many proteins are involved in DNA replication in Ecoli and is it more or less than in humans?
about 20 which is more than what is used for humans
What is DUE? (in Ecoli)
a 13 base pair sequence that unwinds the DNA to start the process of replication
What does it mean when you see GATC during DNA replication in bacteria?
means mismatch repair is taking place at the methylation site which tells us that replication has been initiated at this site (THIS WILL ONLY HAPPEN ONCE)
What are R region (R1-R5) seen in DNA replication in Ecoli?
a DnaA protein binding site that has five regions with a 9 base pair sequence
What happens at the R regions (R1-R5)?
DnaA protein binds at these 5 sites and causes DUE to open up into single strand portions
What is DnaA?
A protein required to initiate replication at the E. Coli origin
What are the 8 proteins required to initiate replication at the E. Coli origin?
DnaA DnaB (helicase) DnaC HU DnaG (primase) SSB DNA gyrase Dam methylase
What does DnaB (helicase) do?
unwinds DNA to expose a single strand template
What does DnaC do?
is a helper for DnaB
What is HU?
histonelike protein (remember bacteria doesn’t have true histones)
What does DnaG (primase) do?
synthesizes RNA primers
What is SSB and what does it do?
single strand binding protein- binds single stranded DNA
What does DNA gyrase do?
relieves torsional strain generated by DNA unwinding
What does Dam methylase do?
involved in mismatch repair system - will tag the parent strand and tell the fixing mechanism which strand is defective and needs to be fixed
Why does the DNA unwind at the DUE sequence?
it has a lot of A-T bonds which are not very strong so it is easier to twist open here
What 2 things bind to form an initial complex?
dnaA and oriC
What is the prepriming complex?
once DnaA is established DnaB is brought in with DnaC to stabilize the unwound DUE elements
What is single stranded DNA stabilized by?
single strand binding proteins (SSBs)
Do all the steps in the initiation of replication need energy?
yes all need ATP
What do all synthesizing enzymes require?
a primer
What is a problem for initiation of DNA replication?
primers can only add to the 3’ end of an existing strand and cannot initiate a new strand
What 2 things bind to form the replisome?
DNA polymerase III and primosome
What do replisomes do?
move around chromosomes (bidirectional replication from a fixed origin)
What is primase?
a specialized RNA polymerase that synthesizes short 15 nucleotide stretch of RNA on DNA template which remains base paired
What enzyme creates the primer for DNA polymerase?
primase
Primase binds to DnaB and DnaC on the DNA template (leading strand ) other proteins add on to form a complex called what?
primosome
What is a problem for DNA elongation?
all DNA polymerases add bases to the 3’ end , has NEVER been found to add bases to the 5’end
What is the new daughter strand called?
leading strand
What is associated with the lagging strand?
okazaki fragments
Why is the lagging strand made in fragments?
it is synthesized in short stretches of several hundred to a few thousand bases (okazaki fragments) because of the orientation required by DNA polymerase
What happens in front of the replication fork?
positive supercoiling
What molecules are in charge of relieving the coiling at the replication forks?
DNA gyrase and topoisomerase I
they maintain the chromosome negatively supercoiled
What happens if DNA gyrase is not working?
positive supercoiling stays intact and replication will stop
What are DNA gyrase inhibitors?
antibiotics that inhibit DNA gyrase and subsequently stop replication
How is elongation initiated?
by forming a loop of the lagging strand template on the replisome
(loops around so it is in the same direction as the leading strand) - just a small section
What begins the synthesis of the lagging strand?
second DNA polymerase III molecule that binds to a primer
What is found on the 5’ end of each okazaki fragment?
an RNA primer
Are okazaki fragments covalently linked to each other?
no
What repairs okazaki fragments and produce a continuous DNA lagging strand?
DNA polymerase I and DNA ligase
What type of enzyme activity does DNA polymerase I have?
5’ to 3’ exonuclease activity
How is RNA removed from the 5’ end of the okazaki fragment?
by DNA polymerase I and then DNA ligase covalently seals the gaps in the backbone
What allows the two double helices to separate? (circular arrangement -still talking about ecoli)
toposisomerases
How is DNA replication terminated?
replication forks meet (not much else is really known)
How many polymerases are used in eukaryote DNA replication?
at least 5 (1 in mitochondria)
Is replication initiated at multiple origins or one origin in eukaryotic DNA replication?
multiple origins (roughly every 40-100kb) otherwise chromosome replication would take several days
What are some difference in DNA replication of eukaryotes and Ecoli?
eukaryotes have linear chromosomes ecoli has a double stranded circle eukaryotes have multiple origins and ecoli only has one
What are the 5 DNA polymerases in eukaryotes?
alpha beta gamma delta epsilon
What does alpha do?
it is a primase
What two eukaryotic polymerases are involved in nuclear replication, nucleotide excision repair (NER) and mismatch repair (MMR)?
delta and epsilon
What does beta do?
mostly used for repair (base excision repair)
What does gamma do?
involved in mitochondrial replication
Why is linear chromosomal DNA a problem for replicating the ends of the DNA molecule in eukaryotes?
the end of the lagging strand that has the primer cannot be fixed so it would just cause shorter and shorter chromosomes in each replication cycle
What are telomeres and what do they do?
telomeres are at the ends of eukaryotic chromosomes and they allow linear molecules to be replicated without loss of information
How are telomeres synthesized?
without a DNA template by the enzyme telomerase
What is telomerase?
a specialized reverse transcriptase that has an RNA component to serve as a template for the synthesis of a repetitive GT rich DNA sequence
also attaches chromosomes to the nuclear envelope
Can priming occur in the telomere? Can coding of DNA occur here?
priming can occur here but coding of DNA cannot
What do telomeres prevent?
undesirable fusion of chromosomes
avoids aberrant recombination
What does it mean for telomeres to serve as a mitotic clock?
interesting for cancer research - if telomerase is blocked it can stop replication
What is senescence?
when cells don’t die they are not replicating but they are just sitting there (shorter telomeres induce this and apoptosis)
Is telomerase active in germ cells, stem cells and tumors?
yes