Lecture 3 Flashcards
what must have low mutiation rate to maintain speciese?
germ cells
Most errors are corrected by
proofreading and DNA repair
post-relication repair mechanism
only 1 mistake every 10^9 base pair, 3 nucleotides every cell division
What needs to have low mutation rates to avoid uncontrolled proliferation/ cancer?
somatic cells
What synthesize DNA by catalyzing
DNA plymerase
the following reaction is catalyzed
DNAn residues + dNTP —> (DNAn+1 residues + P2O7^4-
How is the new DNA strand made?
template directed
What must be seperated for replication?
two parental strands
What does DNA replications require? 4 thins
dATP
dGTP
dCTP
dTTP
What needds a primer with a 3’ -OH to begin?
DNA polymerase
DNA feeds through the …. during replication
polemerase
Both strands are …. replicated
simultaneously
DNa plymerase can only synthesize DNA in the
5’ to 3’ direction
synthesized continualsly
leading strand
synthesized in segemnts
lagging strand
The enzyme tightens its “fingers” around the active site…
which is easiest if the correct base pair is there
exonucleolytic proofreading
takes place immediatly after incorrect bases is added
DNA plymerease requries a perfectly paired 3; terminus
3’ to 5’ exonuclease clips off unpaired residues at 3’ primer terminus
5 to 3 allows efficent error correction
when a high energy bond is cleaved, provides energy for plymerization of that bond.
if it went the other direction, there would not be sufficent energy
Laggin Strand Synthesis
backstitching process
DNA PRIMASE synthesizes an 10nt long RNA primer to rpime DNA synthesis.
RNA primer is erased by RNAseH and replaced with DNA; DNA ligase joins the ends
What joins the ends?
DNA ligase
what does DNA helicase do?
unwinds DNA
protein with 6 identical subunits uses AT hydrolyses
cuases confomational change
helps stabalize DNA, prevents hairpins, base pairing, allows DNA polimerase to still read and put in new
single- stranded DNA binding protein
keep DNA polymerase on DNA when moving; releses when double stranded DNA is encountered
sliding clamp
what pust the sliding clamp onto a primer template junction??
clamp loader
with laggins strand clamp loader is always present since it is procuding DNA in segments and the sliding clamp needs to continually be put bakc on
mismatch repair
MutS binds to mismatch;
Mut: scans for the nick and triggers degradation of nicked strand
How does it know if it is fixing the new strand or the old strnad?
it fixes the non methalated strand, which is the new strand… this is E coli
Type 1 Topoisomerases
makes a single cut on one strand in the phosphodiester bond
thermodynamically favorable process
allows to remove tention and spin around
resealing is rapid
Type II topoisomerase
makes double stranded break in DNA
1. brekas on double stranded helix reversibly to create gate
2. causes second strand to pass through
3. reseals break and dissociates
can seperate “decatenate” 2 interlocked DNA circules
can prevent severe tangling problems
Replication origins
rich in A-T based pairs
bacteria-inition is only pt, only when sufficent nutrients, refractory periord for new strands to be metholated, 2 replication forks do all of it
euarkyotes- need more than 1 origin,
Initiation of DNA replication in bacteria
initiator protein binds to specific site in ORI froming complex
Complex attracts DNA helicase + helicase loader
helicase is placed around a SS DNA exposed by assembly of complex
helicase loader remains engaged until helicase properly loaded
Helicase unwinds DNA so primase can make RNA primer on leading strand; remaining proteins assemble to create 2 replication formks w complexes moving in opposite direction w respect to the ORI
Eukaryotic DNA replication
only occurs during S phase, 8 hours
done in clusters 20-80
heterochromatin is late replicating
Origin of replication Yeast eukaryote
min requirements 3
binding site for ORC
A-T rich stretch for easy unwinding
binding site for proteins that thelp attract ORC(origin of replication complex)
ORC interaction w/ ORI persists
throughout cell cycle
protein that beind to from a ….
prereplicative complex and regulates origin acitivity
done by helicase and helicase loading proteins, Cdc6 and Cdt1
Regulation of OR in Eukaryotes
In S phase,
activated Cdks lead to
- dissociation of helicase loading proteins
- activation of helicase
- unwinding of DNA
- loading of DNA polymerase, etc.
Regulation in eukaryotes of ORC
prevent assembly of new ORC until next M phase resets cycle
single chance to form in G1 when Cdk activity is low
second window for pre-replicative complexes to be activated and disassembles in S phase when Cdks activity is high
ORI function depends critically on distanct sequences
also affects transcription
global effect of decondensing chromatin structure
DNA requires not only DNA but
synthesis and assembly of new proteins
histone proteins are synthesised in what phase
S phase
What is needed to destabilize DNA histone interface?
chromatin remodeling proteins
As replication fork passes through chromatin, histone octamer breaks into;
- an H3-H4 tetramer, distributed randombly to daughter duplexes
- 2 H2A-H2B dimers which are released from the DNA
H2A/H2B dimers are 1/2 old and 1/2 new
they are added at random to complete complex
What does the addition of hisones require?
Histone chaperones (chromatin assembly factors)
What is the sliding clamp called that directs to DNA?
PCNA
Patterns of Histone modification can be inherited
some contain only parental histones, some only new but most are hybrids of new and old.
parental patterns of histone modification are spread through reader-writer complexes
epigentic inheritance responsibility
End replication problem on lagging strand:
no place for RNA primer
bacteria have cirular genome
eukakaryores have telomeres
What is the special sequence at the end of eache chromosome
GGGTTA
What enzyme relenishes these sequences by elongating parental strand in 5’ to 3’ direction using an RNA template on the enzyme
telomerase it’s a protein and RNA combination
What completes the telomerase replication of laggins strands?
DNA plymerase, using extension as template This mechanism(plus a 5' nuclease) ensures 3' end is longer, leaving a protruding SS end that loops back and tucks into the repeat
structures protect ends and distinguishes them from broken ones that need to be repaired
T-loops
What cells have full complement of telomere repeats at birth?
somatic cells
What retain full telomerase activity?
stem cells
Each chromosome end in a given cell contains variable # of telomere repeats depending on age
repeats are lost each genreation due to insufficient telomerase acitivity
after many generations, daughter cells will have defective chromosomes and stop dividing; in this way the cell’s lifetime is regulated to guard against cancer
replicative senescence
may cause againg
How many times do human fibroblasts divide before undergoing replicative senescence
60 times
dyskeratosis congenita
carry mutant telomerase RNA gene
