replication 1 Flashcards
what phase of cell cycle is DNA replication
s-phase
what is the name of the region of a chromosome/plasmid were replication initiates
origin (ori)
how many origins of replication do prokaryotes and eukaryotes have?
prokaryotes have one
eurkarytoes have multiple/chromosome
what is a replicon
a unit of DNA that is replicated from one origin of replication
how often/cell cycle does a replicon work?
once
depending on cell type and age human chromosomes have how many replicons?
10,000 - 100,000
BrdU is an analog to what
thymine
what is a foci
an area of DNA with up to several hundred replication forks
define semiconservative DNA replication
Each strand acts as a template for the synthesis of a new DNA molecule by the sequential addition of complementary base pairs, generating a complementary base sequence
how was it discovered DNA replication forks are bidirectional?
cells were grown in lightly radiolabeled thymine to lightly label all DNA then for a brief time grown in heavily labeled thymine to heavy label it, the rep fork was dense, chromosomes were imaged and label was seen equally at both replication forks
exception to bidirectional replication?
colE1 plasmid in e. coli. was discover by electron microscopy of restriction enzyme treated plasmid, after linearization the rep bubble was same distance from cut each time
all DNA polymerases make DNA in what direction
5’ –> 3’
describe pulse chase experiment that discovered okazaki fragmentss
e.coli were infected with a bacteriaphage, the phage duplicating, while duplicating different times pulses of radioactivity, the shorter pulses = shorter DNA fragment, = okazaki fragments
what is a primer that allows DNA synthesis adding?
a 3’-OH
nick in preceding DNA, and RNA primer, or a DNA binding protein with covalent nucleotide link can serve as primers
most common replication structure in bacteria, what type of primer?
theta mode, RNA
explain rolling circle replication
circular DNA, on stand nicked, 3’ end is extended/5’ end is displaced
DNA polymerase I in e.coli 3 distinct activities, what are they/what do they do
5’->3’ polymerase replaces removed primer with new DNA (nick translation)
3’->5’ exonuclease proofreading (“klenow, finger/palm)
5’->3’ exonuclease removes RNA primer
why was studying DNA polymerase III done in a conditional mutant?
zero cell proliferation if no DNA poly III, needed to study under restrictive and permissive conditions
important subunits of DNA polymerase III holoenzyme (3)
alpha, DNA polymerase
epsilon, 3’ -> 5’ exonuclease
beta, sliding clamp
what protein is the initiator of chromosomal replication?
DNA A
what is the motif that is recognized to begin e.coli replication
DNA A box in the oriC
throughout the cell cycle/prior to replication initiation what proteins are bound to e.coli ORC?
the high affinity DnaA boxes R1, R2, R3 bound by DnaA and Fis (repressor) binds inbetween
what proteins bind do ORC during replication initiation of e.coli to form prereplication complex
DnaA is able to bind moderate and low affinity DnaA boxes (tau and I sites)
Fis (repressor) is displaced by IHF
How is DNA unwound by prereplication complex in e.coli?
tortional stress from DnaA protein monomers
name of protein that binds DnaA to accelerate/coordinate cell cycle initiation
DiaA (DnaA initiator associating protein)
after unwinding of AT-rich oriC region DnaA recuits what
two DnaB-DnaC complex
B = helicase (hexameric, unwind DNA at rep fork)
C = helicase loader, bring DnaB to the DNA then dissociates
name of protein that bind DNA after unwinding and it’s purpose
single strand binding protein (SSB) prevents reannealing
what are the proteins recruited by DnaB to make RNA primers
DnaG primase and DNA pol III holoenzyme (extends primers)
how does methylation prevent over replication of ecoli DNA
Dam (DNA adenine methyltransferase) methylates adenines in a palindromic OriC sequence, the protein SeqA binds the hemimethylated areas and inhibits formation of new initiation complexes
as DnaA poorly recognizes hemimethylated DNA can’t initiate replication
How does dnaA regulate itself
DnaA protein functions as its own repressor
after chromosomes double DnaA will be sequestered elsewhere in the genome
