DNA Replication Flashcards
speed of DNA replication
fast; takes a few hours to replicate 3.3 billion bp of human DNA
mistakes in DNA replication
have 3’ to 5’ exonuclease activity that enables to proofread and fix mistakes; DNA repair proteins that can replace incorrectly placed nucleotide; these features bring error rate to 1 per 1 million nucleotides
conservative replication
original DNA molecule stays intact and completely new molecule is synthesized
dispersive replication
original DNA molecule gets fragmented & fragments used as templates for synthesis of new DNA then the chromosomes get reassembled; resultant chromosomes have old & new DNA scattered throughout both strands
semiconservative replication
original DNA molecule unwinds & each strand is used as template for synthesis of new strand; resultant chromosomes have one old strand and one new strand
Meselson and Stahl
demonstrated that DNA replication is semiconservative
Meselson and Stahl experiment
grew DNA molecules in nitrogen isotopes of different weights: after 2 rounds of some DNA was light and some was intermediate suggesting semiconservative replication
Taylor
confirmed semiconservative replication in eukaryotes
Taylor experiment
bean root tips & radioactive tritium; one round mitosis: both sister chromatids labelled; 2 rounds of mitosis: only one sister chromatid from each chromosome labelled
theta replication
in organisms w circular chromosomes (bacteria); double helix unwinds at single origin of replication creating replication bubble w replication fork at each end; replication proceeds in both directions
rolling circle replication
viruses & plasmids; one DNA strand cut to 5’ phosphate end and 3’ OH end; new nts added to 3’ end using intact other strand as template as new DNA strand elongates it pushes 5’ end of cut strand off other strand; old strand cleaved off other strand & circularizes and is used as template for replication ending w two circular DNA molecules
replicating linear chromosomes
many origins of replication - replication in both directions; each replication bubble produces one replicon
order of events in DNA replication
initiator protein/complex recognizes origin of replication; helicase unwinds double helix separating 2 DNA strands; single strand binding proteins keep 2 DNA strands from reannealing; topoisomerase nicks one strand of DNA in regions just outside rep forks to release tension caused by unwind tightly coiled DNA mol - so cell can cont to open double helix and replicate all its DNA; primase lays down primer that contain RNA nucleotides; DNA polymerase uses DNA strand as template & synthesizes complementary strand; DNA polymerase removes RNA primers and replaces them w DNA nucelotides; DNA ligase knits all pieces together: DNA frags that replaced RNA primers, stretches of DNA between primers, Okazaki fragments
origin of replication
OriC; has characteristic repetitive seq (rich in A/Ts) that varies a bit from species to species
origin recognition complex (ORC)
in eukaryotes ORC binds to origin of rep and causes replication licensing factor (RLF) to bind there as well; RLF attaches to origin of replication and unwinds a little of the helix allowing DNA rep to begin
DNA helicase
opens helix up to expose the bases which creates replication bubble w replication forks at either end
single stand binding proteins
hold helix open
topoisomerase I
breaks DNA strand outside open region in eukaryotes allowing strands to pass through each other then close the break; if tension doesn’t get relieve helix will not be able to open up very far & most of the DNA will not be accessible to replication machinery
4-quinoline antibiotics
(ex: ciprofloxacin) kill bacteria by inhibiting gyrase the topoisomerase used by bacteria
DnaG
lays down stretch of 10-12 RNA nucleotides (ribonucleotides) in bacteria
DNA polymerase alpha
lays down primer in eukaryotes; primer is mad of RNA & DNA - consists of approx 10-12 ribonucleotides plus approx 23-30 deoxyribonucleotides
DNA polymerase delta
replicates lagging strand in eukaryotes
DNA polymerase epsilon
replicates leading strand in eukaryotes (DNA polymerase III does this in bacteria)
replication protein A (RPA) & DNA polymerase delta
remove primers in eukaryotes; RPA unwinds DNA/RNA hybrid causing one end of primer to stick out like loose flap and binds to loose flap of RNA to keep it from reannealing
endonuclease
recruits by RPA to cut at other end of primer removing primer DNA polymerase delta then fills in gap w DNA nucleotides (DNA polymerase I does this in prokaryotes)
DNA ligase
knits diff frags together: DNA frags that replaced RNA primers, stretches of DNA between primers, Okazaki frags
leading strand
replicated continuously to the end of chromosomes or to next rep fork whichever comes first; DNA polymerase can only add to 3’ end
lagging strand
synthesized in fragments of approx 1500 nucleotides (Okazaki fragments)
eukaryotes & telomeres
cannot replicate telomeres; length of telomeres regulates lifespan of eu cell = when telomeres reach certain length cell goes into apoptosis
telomerase
RNA protein complex that fills in telomeres; in human only active in few cell types ex: gametes or B cells of immune system; RNA portion of telomerase binds to DNA & acts as template for new synthesis; protein portion synthesizes new DNA to fill in telomere
telomerase inhibitors
help battle cancers that are caused by gain of function mutation in telomerase
Werner syndrome
premature aging syndrome; defect in RecQ helicase: enzyme that is essential for telomere replication
histone chaperone proteins
help assemble histones into 8 histone cluster
chromatin assembly factor 1 (CAF1)
helps assemble DNA back into proper configuration
replication licensing factor (RLF or MCM)
to avoid duplicate rep; contains several proteins including helicase and protein geminim; RLF attaches to origin of replication & unwinds little of the helix allowing DNA replication to begin
geminim
present on origin of rep prevents another RLF from attaching; degraded in G1 so DNA rep can reoccur in next S phase
ter sites
signal termination of replication
tus (terminus utilization substance) protein
binds to one of the ter sites and stops one of the rep forks allowing other fork to come and meet it
topoisomerase II
forms double stranded break in DNA allowing 2 circular DNAs to become untangled so they can migrate into 2 daughter cells when cell splits
avoiding duplicate rep in prokaryotes
tus protein and ter sites
