Module 5 lecture 5-9 Flashcards
in watson and cricks report in the journal they stated
the pairing proposed does suggest a possible copying mechanism for genetic material; replication
watson and crick favored what model of DNA Replication
Semi-conservative
what models where there of DNA replication
semi-conservative model, conservative model, dispersive model
Semiconservative replication
one strand is used as a copy for 2 new molecules
conservative replication
he parental double helix remains
intact; both strands of the daughter double helix are newly
synthesized
dispersive replication
At completion, both strands of both
double helices contain both original and newly synthesized
material.
mehelson stahl experiment was used to determine
which type of replication occured
describe mehelson stahl experiment
-started out with “normal” DNA
-They grew it for one
generation with the heavy
isotope 15N instead of normal
14N
-
weight differences between N15 and N14 allowed tracking
N15 able to track
What DNA-“weight” would
you expect for any of the
three suggested models in
the first generation
Conservative: Two distinct bands (heavy and light).
Semi-Conservative: One intermediate band.
Dispersive: One intermediate band
mehelson stahl experiment control
e. coli grown in N14-> dna extracted from cells-> centrifuge
results N14N14 single band towards top of tube
mehelson stahl experiment first part
e. coli grown for many generation in N15 medium->Extract DNA->Centrifuge
results: 1 band of dna N15 towards the bottom
mehelson stahl experiment second part
N15 cells transfered to N14 medium->cells replicate to form first generation of daughter cells-> extract DNA-> centrifuge
Results: single band in middle of tube in between n15 and N14. meant DNA strand had both n15 and N14
mehelson stahl experiment third part
cells replicate to form second generation of daughter cells-> extract DNA-> centrifuge
results: band of hybrid N15N14, and band of N14N14
After growing Escherichia coli for several generations in a
medium containing 15N, they found that the DNA of the cells
was heavier than normal because
N15 atoms are heavier
what determines where it accumulates in the tube after centrifuge
density of DNA
semiconservative replication detailed model shows
-original double helix
-strands seperate
-complementary bases align opposite templates (A-T, C-G)
-enzymes link sugar phosphate backbone of aligned nucleotide into continuous strand
end result of semiconservative replication
2 molecules of DNA each has old strand and new strand. if layed on top of each other they should be identical.
Complementary base pairing produces
semi-conservative
replication
summary of semiconservative replication
-Double helix unwinds
-Strands separate
-Each strand acts as template for new strand
- Complementary base pairing ensures that T signals addition of A on new strand, and G signals addition of C
-Two daughter helices produced after replication
New daughter strands have to be synthesized from
free nucleotides
5-3 synthesis is _____ then 3-5
easier
in 5-3 there is
a bond made between hydroxal group on 3 carbon and incoming nucleotide phosphate group
DNA polymerases only works
5’ to 3’
DNA polymerase enzyme can only connect a
new nucleotide
to a free third carbon atom of a preceding nucleotide
what different abou RNA as appose to DNA
RNA polymerases can start from scratch
in dna replication there are _______ and ______ strands
leading and lagging
Leading strand is made
continuously in 5’->3’ direction following the moving replication
fork
in leading strand The initial free 3’ end is provided by
a short RNA prime
primer is made by ________
Primase
primase=
rna polymerase
lagging strand is made
discontinuously in form of smaller fragments
what are the smaller fragments in Lagging strand
1000 nucleotides and okazaki fragments
in lagging strand there are _____ fragments
shorter
Each shorter fragment is started by an
RNA PRIMER
how many okazaki fragments are there
2
After two Okazaki fragments are finished they are connected by a
DNA Ligase
what are some key enzymes involved in dna replication (6)
DNA helicase
Pol III
Pol I
Primase
Ligase
Dna helicase
unwinds double helix
polymerase III
produces new strands of complementary DNA
Polymerase I
fills in gaps between newly synthesized Okazaki
segments
Primase
creates RNA primers to initiate synthesis
ligase
welds together Okazaki fragments
how many key enzymes are involved in dna replication
6
Single-stranded binding proteins
keep helix open
primary enzyme of dna synthesis
POL III
what enzymes re implicated in DNA repair
Pol I and Pol II
All DNA polymerase (I, II, III) have
3-5 prime exonuclease activity
whats special about DNA pol i as apposed to Pol I and II.
Polymerase I participates in 5-3 prime exonucelase activity.
3-5 prime exonuclease activity is involved in
proofreading
DNA replication starts at
origin of replication
replication bubble made using 2
replication forks
lagging strand is tied together by
ligase enzyme
in eukaryotes okazaki fragments are smaller. how small?
150 nucleotides
how many “classic” DNA polymerases are there
5
what are the 5 “classic” DNA polymerases
Pol a
pol s
pol e
pol y
pol b
pol a (alpha)
extends RNA primer with 20 nucleotides
pol s
main polymerase, when reaching next Okazaki fragment it displaces fragment and keeps synthesizing DNA
what is the main polymerase
pol s
Pol y
replicates mitochondrial genome
pol B (beta)
does dna repair
pol y, s, e have
exonucelase activity and (proofreading)
in eukarotes Replication forks move in
opposite directions
is the replication fork moves in opposite directions then
Replication is bidirectional
Eukaryotic chromosomes have several hundred
origins of replication
In circular chromosomes, such as E. coli, there is only how many ORIC
one origin of replication
in linear chromosomes, telomeres ensure the
maintenance and accurate replication of chromosome ends
Unwinding of chromosomes causes
supercoiling
Errors during replication are ____
rare
after errors during replication are made they are
immidientely corrected
Enzymes repair ________ to DNA
chemical change
Complementary strands are ______
redundant
telomeres solve the
end problem
unwinding of chromosomes at one location causes another location to
wind tightly (supercoil)
solution to supercoil is
topoisomerases
topoisomerase works by
cutting dna, rotate strands so not winded, then strand is rejoined by ligase.
topoisomerase is in
eukaryotes and prokaryotes
important topoisomerase in bacteria is known as
girase
since complementay strands are redundant
basis to repair errors correctly either during or after replication
what generates diversity
mutations and New combinations of already existing alleles
how can New combinations of already existing alleles occur (3)
-Independent assortment of homologous chromosomes
-Crossing-over
-recombination
Molecular Mechanism of Crossing-over first step is
initial cut, by spo11
what enzyme is involved in cutting strand of chromatids
spo11
non sister chromatids involved in
crossing over
Molecular Mechanism of Crossing-over second step
exonuclease chew away from intial cuts and 3 prim single stranded tails are formed
one of the tails from cut made will ______ another chromatid
invade, and takes its place
when chromatid is invaded and strands place is taken the previous strand that was there before is
displaced
displacement of strand during strand invasion forms
D loop
Molecular Mechanism of Crossing-over third step
formation of holiday junction
formation of holiday junction
2 crosses between strands of 2 different chromatids
holiday junctions can
migrate.
when holiday junctions migrate we have a
heteroduplex (in between holiday junctions)
holiday junction migration moves towards
holiday junctions
Whether Crossing over Occurs Depends on
the Resolution of Both Holliday Junctions
if resolution of intermediates is the same there will be ______. if it is the same there is_____
gene conversion; crossing over
in heteroduplex there are
2 chromosomes, 4 chromatids, 4 alleles
after heteroduplex form and repair there are
Mismatch repair no. 1 and 2
Mismatch repair no. 3 and 4
Mismatch repair no. 3 and 4
leads to allele frequency changes
Mismatch repair no. 1 and 2
regenerate original allele
combinations
how many chromatids are involved in crossovers
2
what happens to the 2 unused chromatids in crossovers
remain same
If original was AA BB after mismatch repair, no. 3 and 4 result in
genotypes ABBB or AAAB
Allele ratios change from 2:2 to 3:1 or 1:3
when allelic changes occur as a result of heteroduplex resolution we get
gene conversion
What Happens in Heteroduplexes?
-If the two strands are not identical we end up with a base pair mismatch in the heteroduplex
The mismatch will be fixed by _______
cellular enzymes
how do cellular enzymes fix mismatches
cut out the wrong base and replace it
But how does the enzyme know which base has to be replaced
enzymes recognize pairing isnt correct
how does heteroduplex work
allele A- ATATCG. allele C- ATCTCG
AATAGC TAGAGC
after reombination what would happened
the strands are similar so there could be a mixmatch in the dna bases
mismatches can be repairs to
A/A or B/B repairs. original A allele. and B/B means original B allele
heteroduplex can make
mismatch pairs.
