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lec 10 and recap
what is good about anti mutators
has high quality for a slower time
takes long but has high fidelity
what is mutogenesis used for
to induce random mutoations into a population of cells
what are cells with higher than normal mutation rated called
the mutated strains
that are cells with lower mutation rates
anti mutator strains
what is the first dna proof reading step carried out by
dna polymerase and occurs before the new nucleotide is added to the growing chain
correct nucleotide fit with the complementary base has higher affinity for the moving polymerase compared to the incorrect nucleotide
active site of polymerase only accommodates for the right size pairs
after nucleotide binding ( before formation of a covalent bond) the polymerase undergo a conformational change ( closing fingers) and incorrectly pound nucleotides can be rejected
what is a con of ultra high fidetlity
thates a significant amount of time
causes the trade off of speed or accuracey in the mutated strain an dthe antimutated strain
what helps direct/ coordinate phosphodiester formation
tyrosine and its rings, positive tyrosine and negative phosphates therfreo bind
what is the second proof reading step
mismatch repair
mediated 3’-5’ exonuclease activity of polymerase
actively removes misincorporated nucleotides
if the wrong is added and continues that is a mismatch and can be detected and repaired
when it is not the right since it stops, goes back. chews off with exo nuclease on that last nucleotide
it is methyl directed and used to mark new form old and determine directionally the repair correctly
what is the final proofreading step
strand detection of mismatch repair mechanisms to further lower error rates in dna replication
what is mutS for
detects distortion of the dna helix and binds to a mismatched base pair
what is mutL for
binds the mutS-dna complex and activates mutH at near by methylated GATC to introduce a nick on the unmethylated strand of the dna duplex
once bound what does mutS and mutL do
can nearby dna for a nick, once detected mutl recruits helicase to separate dna strands and exonuclease to degrade the nicked strand all the way back pass the mismatch
what does dna pol 3 do during mismatch repair
comes in and fills in the gap using methylated strand as template
which strand will be methylated after replication
only the parental strand
what does mut h do
Activated by MuteL and detects the presence of a methylated nucleotide
scans for methylated gatc on either side of a mismatch
why was molecular assy developed/ what does it involve
involves
creating a double stranded plasmid with a mismatch
plasmid contains restriction enzyme recognition sites engineered for resistance
incubation of cells
repaired plasmid is tested for the restriction enzyme sites
if restored the plasmid will be sensitive to the restriction enzyme confirming that the mismatch has been repaired
what phase does dna replication occur in
DNA replication only occurs during the S (synthesis) phase of the cell cycle.
difference between eukaryotic and prokaryotes in relation to genomes
eukaryotic cells have a much larger genome and are chromatized, linear chromosomes with many origins for replication
what are the cell cycle phases
G0, G1, S, G2, and M S phase
which has a slower rate of replication
eukaryotes are slower likely because of chromatin
what is ARS
autonomously replication sequences are origins in yeast that are well defined
how are origins found in eukaryotes
licensing factors such as origin recognition complex,
they mark potential origins during g1 and later additional proteins, mcm2-7 complex and helicase are recruited to initiate replication
what do mcm2-7 proteins do
provide helicase activity for dna synthesis and loading of these proteins confers on the origin to fire in s phase
what is the protein needed for dna synthesis
the action of 2 protein kinases
why is protein kinases needed for dna synthesis
triggers the association of additional proteins with the origin
during the process of initiation dna polymerase are also recruited at the start
where do mcm2-7 proteins go during replication
they move away from the origin and the pre replicative complexes get blocked
this ensures that origins can only fire a single time per cell cycle
what does pol a do
primase + another dna polymerase activiy, it synthesizes both rna and dna primers
what synthesizes the leading strand
pol e
what synthesizes the lagging in strand
alternates between pol a and delta
how is high fidelity mainteined
base pairing, primer selection, proofreading
why does chromatin cause problems/ slow it down
chromatin needs to be disassembles ahead of the replication for and reasspemple nucleosomes post dna replication
pcna directly binds to chormatin remodeling complexes such as caf 1
then nucleosomes are redistributed to new strands
which pol have high fidelity
alpha delta, epsilon
what is the end replication problem
. Every time DNA replicates, the very end of linear chromosomes cannot be replicated due to the absence of a primer for DNA synthesis.
what protective structure helps agains the end replication problem
telomeres
what are telomeres
consist of repeating sequences like TTAGGG in humans.
They exist at the ends of chromosomes and include both double-stranded and single-stranded regions.
The single-stranded overhang is G-rich and extends beyond the double-stranded portion.
why are telomeres important
they preserve chromosome integrity
what is telomere length linked to
preventing cell aging d preserving genetic material
what does firdelity refer to
the accuracy of copying dna to prevent accumulation of mutations during cell division
overall facts for prokaryotic dna replication
occurs in cytoplasm
single region of origin
dnag makes rna primer
pol3= main replicative polymerase
rna primer removed by rnase h
replicated naked dna
okazaki fragments are 1000-2000 nts in length
replicates at 750-1000 per second
overall facts for eukaryotic replication
occurs in nucleus
many origins of replication
pola makes rna and dna primer
pol delta synthesizes lagging strand pole synthesizes leading strand
rna primer removed by rnase h1 and fen1
replicates dna in chromatin context
okazaki fragments are 100-200nts
replicates at 50-100 nts per second
