DNA Replication Flashcards
G1 phase
- period of cell growth
- many structural components and metabolic enzymes are synthesized here
- towards the end DNA polymerases and other replication enzymes are synthesized
G2 phase
- checkpoint that checks to make sure cells are ready to divide
- prevents cells from entering mitosis with DNA damages since last division, providing opportunity for DNA repair and stopping proliferation of damaged cells
- -some synthesis of cell materials occurs here, (tubulin monomers for microtubule assembly)
- synthesis of wall & membrane components
general characteristics of DNA replication
- chemically unidirectional (5’ -> 3’)
- spatially bidirectional (2 forks at once in opposite directions)
- semi-conservative
- semi discontinuous
semi-discontinuous
DNA polymerase can’t stay on DNA strand the whole time without falling off and new polymerase coming on
origin of replication
the region of DNA that first separates and replication begins
bi-directional replication
DNA replication proceeds in both directions away from origin of replication
replication fork
point of separation of double-stranded DNA at which incorporation of nucleotides occurs during DNA replication
semi-conservative replication
where each separated polynucleotide strand serves as a template for the synthesis of a single new complementary strand
okazaki fragments
- the name given to discontinuous fragments of DNA synthesized in lagging strand
- 100-200 nucleotides in eukaryotes
- 1000-2000 nucleotides in prokaryotes
overview of DNA replication in E. coli
- initial unwinding, separation, and stabilization of duplex DNA (several proteins)
- primer synthesis (primase- RNA template)
- DNA synthesis (DNA polymerase)
- Replace RNA primers with DNA
- seal gaps between okazaki fragments on lagging strand (ligase)
- termination of synthesis (“ter” protein, telomerase)
initiation
-unwind and stabilize duplex DNA to form replication fork
initiation factors
DnaA proteins bind to origin of replication
helicase
DnaB protein catalyzes ATP-deendent unwinding of duplex DNA
topoisomerases
- prevent supercoiling & tangling of DNA during unwinding
- bind ahead of replication fork, nick supercoiling DNA, releases stress by allowing uncoiling
formation of replication fork
- 20-40 DnaA proteins bind to oriC sequence forming nucleosome-like structure
- localized melting causes a small segment of DNA to open up
- DnaB (helicase) enters oriC region and unwinds DNA
- single stranded binding proteins attach to keep strands apart and protect against nucleases
oriC
origin sequence, where initiation of replication begins
-contains four 9 base pair repeats
primase
- an RNA polymerase synthesizes a short RNA primer
- DNA polymerase CANT initiate DNA synthesis-can only add nucleotides to the end of a chain that is base-paired with template strand
- RNA polymerase can initiate synthesis without a primer
DNA polymerase I function
- fills in gaps
- repairs miss-matched pairs
- replaces primer RNA during replication with DNA
DNA polymerase II function
- thought to also be involved in some repair processes
- prevalent during stationary phase
DNA polymerase III function
- the main polymerase of E. coli
- extends RNA-primed chain
DNA ligase
joins DNA (okazaki) fragments
E exonuclease repair function
- enzyme recognizes mispaired bubbles in DNA
- backs up, excises, then polymerase resumes replication
- 3’ to 5’ exonuclease
theta subunit function
unknown
core polymerase composition
alpha, epsilon, and theta subunits
beta subunit
- sliding clamp that helps keep DNA polymerase bound to template during many rounds of nucleotide addition
- converts polymerase from distributive enzyme to progressive enzyme
T proteins
dimerize 2 DNA polymerase III cores
clamp loading complex
responsible for placing doughnut shaped B subunit around DNA template
processivity
frequency with which an enzyme dissociates from template during DNA replication
alpha subunit function
5’ to 3’ polymerase
E. coli replisome components
- DNA polymerase III (dimer)
- primosome (helicase + primase)
- ssbp
- dimer + looped lagging strand allows synthesis in same direction
Eukaryotic replication
-3 polymerases total (some have dual function)
polymerase delta
-lagging strand
polymerase epsilon
leading strand
polymerase alpha
makes primase
P domain
-acts as sliding clamp in eukaryotic replication
eukaryotic replication of linear chromosome
- bidirectional and proceeds from several fixed origins
- forks advance until they meet another fork traveling in the opposite direction
- origins are programmed to initiate replication at fixed times in S phase
- requires that replication origins become active at different times
- chromatin must be dismantled prior to replication fork
telomere
- series of repeated TTAGGG DNA sequences located at the ends of linear eukaryotic chromosomes
- each time a cell divides, some of the telomere is lost due to exonuclease activity
- eventually little/no telomere remains but degradation continues
- cell dies
- typically one telomerase is G rich and the other is C rich.
telomeres and cloning
premature aging and death of animal clots thought to be related to cloning from cells/nuclei with partly reduced telomeres
telomerase
- enzyme that restores telomere sequence
- thought to restore chromosome/cell longevity
- some cancers may be due to over-active telomerase
hayflick’s limit
50 +or- 10
-number of cell divisions on average
problems with synthesis completion on the 5’ end of linear genomes
- excision of RNA primer would leave gap that can’t be filled bc no 3’ primer terminus to extend
- viruses have evolved 3 strategies to overcome
- eukaryotes solve problem using telomeres
termination of DNA replication in prokaryotes
- ter binding proteins bind to ter sites on opposite side of DNA loop in the bacterial chromosome
- inhibits helicase and prevents further progression of replication forks
termination of DNA replication in eukaryotes
- DNA polymerase runs off the ends of DNA
- replication bubbles fuse as polymerases collide
- involves telomeres (sequences at ends of chromosomes that buffer against the loss of critical coding sequences following a round of DNA replication)
retroviruses
- use reverse transcriptase to produce DNA from RNA template
- RNA viruses steal tRNA from host cell to use as primer
- starts to extend primer towards opposite end of viral genome
- Rnase H cuts off portion of viral genome to allow RNA to circularize
- RNA synthase keeps synthesizing circular DNA
- eventually enters host cell’s genome
- cuts somewhere using integrase and incorporates itself
template dna in vitro pcr
provides desired sequence of gene to be expressed
buffer with Mg2+ included in vitro pcr
provides environmental conditions necessary to carry out rxn
dna polymerase (Taq) in vitro pcr
catalyzes dna elongation
dNTPs in vitro pcr
allows DNA polymerase to make copies of template DNA
primers in vitro pcr
hybridize with specific portion of template DNA to initiate synthesis of new strands
