2.2. DNA Replication Flashcards
what are the universal features of replication?
(1) both DNA strands as template
(2) semiconservative mode of replication
(3) bidirectional synthesis
(4) 5’ to 3’ direction of synthesis
(5) specific base pairing
(6) requirement for RNA primer
explain how dna is reproduced by semi-conservative replication
each strand of the double helix serves as a template for a new strand. when the dna replicates, the two strands separate, and new complementary strands are synthesized for each of the original strands. as a result, each of the two new DNA molecules contains one original strand and one newly sythesized strand.
type of replication wherein the parent DNA molecule remains intact. after replication, one completely new double helix is created and the original helix is conserved
conserved replication
type of replication wherein the parental strands are dispersed into two new double helices following replication. hence, each strand consists of both old and new DNA
dispersive replication
discuss the meselson-stahl experiment
(1) e. coli is initially grown in a medium containing the heavy isotope 15N, resulting in DNA labeled with this heavier isotope
(2) the bacteria are then transferred to a medium with the lighter isotope 14N, causing newly synthesized DNA to incorporate the lighter nitrogen
(3) using cesium chloride density gradient centrifugation, DNA is separated based on density, revealing bands corresponding to different generations of DNA replication.
(4) after one generation, DNA showed a single band of intermediate density, which supports semi-conservative replication; each DNA molecule has one old (heavy) strand and one new (light) strand. however, it also reules out conservative replication, in which two distinct bands would occur
(5) after two cell divisions, DNA showed two bands: one intermediate and one lighter. similar results occurred after a third generation, except that the proportion of the lighter band increased. this was again consistent with the interpretation that replication is semiconservative.
what type of synthesis does DNA follow? explain.
bidirectional synthesis : creates two replication forks that move in opposite direction away from the origin of synthesis
formed by the juncture where the two strands separate
replication fork
true or false: in bidirectional synthesis, one replication fork is created.
false. two replication forks are formed in a bidirectional synthesis
segment of DNA that is replicated as a unit from a single origin
replicon
what are the two sites found in a replicon?
ori site and termination site
direction of DNA synthesis
5’ to 3’
a short segment of RNA complementary to DNA
RNA primer
design a 5-bp RNA Primers for the following DNA Sequences:
5’ GGGGCCCTCCTGAACG 3’
3’ CCCCGGGAGGACTTGC 5’
primer 1: 5’ CGUUC 3’
primer 2: 5’ GGGGC 3’
differentiate forward primer from reverse primer in polymerase chain reaction (PCR) primers
forward primer replicates the 3’ to 5’ template, whereas reverse primer replicates the 5’ to 3’ primer
PCR primer: identify forward and reverse primers given these strands
5’ GGGGCCCTCCTGAACG 3’
3’ CCCCGGGAGGACTTGC 5’
forward primer : GGGGC
reverse primer : CGTTC
major DNA replication enzyme in bacteria : replaces supercoils ahead of replisome
enzyme : DNA gyrase
encoding genes : gyrAB
major DNA replication enzyme in bacteria : binds origin of replication to open double helix
enzyme : origin-binding protein
encoding genes : dnaA
major DNA replication enzyme in bacteria : loads helicase at origin
enzyme : helicase loader
encoding genes : dnaC
major DNA replication enzyme in bacteria : unwinds double helix at replication fork
enzyme : helicase
encoding genes : dnaB
major DNA replication enzyme in bacteria : prevents single strands from annealing
enzyme : SSBP
encoding genes : ssb
major DNA replication enzyme in bacteria : primes new strands of DNA
enzyme : primase
encoding genes : dnaG
major DNA replication enzyme in bacteria : main polymerizing enzyme
enzyme : DNA polymerase III
encoding genes : n/a
major DNA replication enzyme in bacteria : loads Pol III onto sliding clamp
enzyme : clamp loader
encoding genes : holA-E
major DNA replication enzyme in bacteria : holds Pol III on DNA
enzyme : sliding clamp
encoding genes : dnaN
major DNA replication enzyme in bacteria : strand elongation
enzyme : polymerase subunit
encoding genes : dnaE
major DNA replication enzyme in bacteria : holds together the two core enzymes for the leading and lagging strands
enzyme : dimerization subunit (tau)
encoding genes : dnaX
major DNA replication enzyme in bacteria : proofreading
enzyme : proofreading subunit
encoding genes : dnaQ
major DNA replication enzyme in bacteria : excises RNA primer and fills in gaps
enzyme : DNA polymerase I
encoding genes : polA
major DNA replication enzyme in bacteria : seals nicks in DNA
enzyme : DNA ligase
encoding genes : ligA, ligB
major DNA replication enzyme in bacteria : binds terminus and blocks progress of the replication fork
enzyme : tus protein
encoding genes : tus
major DNA replication enzyme in bacteria : unlinking of interlocked circles
enzyme : topoisomerase IV
encoding genes : parCE
large replication complex formed by aggregation of replication proteins
replisome
function of DNA polymerase I of e.coli
(1) dna repair
(2) primer removal
(3) filling of gaps from primer removal
function of DNA polymerase III of e.coli
primary replication enzyme
function of DNA polymerase II, IV, V of e.coli
dna repair
true or false : DNA polymerases I, II, and III are not capable of initiating chain synthesis
true
among the three DNA polymerase, which is/are capable of 5’ to 3’ polymerization (addition of nucleotides at the 3’ end)? which is/are capable fo 3’ to 5’ exonuclease activity (proofreading)? which is/are capable of 5’ to 3’ exonuclease activity (removal of RNA primers)?
🔸all three are capable fo 5’ to 3’ polymerization and 3’ to 5’ exonuclease activity
🔸only DNA polymerase I can do 5’ to 3’ exonuclease activity or the removal of RNA primers
active form of DNA polymerase III which is made up of unique polypeptide subunit, such as α, ε, and θ (there are seven other subunits identified; 10 in total)
holoenzyme
a complex made up of the largest sub-unit α, along with subunits ε and θ, which imparts the catalytic function to holoenzyme; elongates polynucleotide chain and proofreads
core enzyme
true or false: in e.coli, each holoenzyme contains two, or possibly three, core enzyme complexes
true
subunit(s) of DNA Pol III holoenzyme responsible for 5’ to 3’ polymerization
α
subunit(s) of DNA Pol III holoenzyme responsible for 3’ to 5’ exonuclease activity
ε
subunit(s) of DNA Pol III holoenzyme responsible for core assembly
θ
subunit(s) of DNA Pol III holoenzyme responsible for loading enzyme on a template, or the (sliding) clamp loader
γ, δ, δ’, χ, ν (known altogether as γ complex)
subunit(s) of DNA Pol III holoenzyme responsible for the sliding clamp factor (processivity factor)
β
subunit(s) of DNA Pol III holoenzyme responsible for dimerizing core complex
τ
how many different DNA polymerases can be found in humans? in mammalian cells?
at least 14; tens of thousands
refers to the strength of the association between the enzymes and its substrate, and thus the length of DNA that is synthesized before the enzyme dissociates from the template
processivity
what are the major eukaryotic DNA polymerases?
(1) Pol α
(2) Pol δ
(3) Pol ε
eukaryotic DNA pol(s) with low processivity; two of its/their four subunits synthesize RNA primers on both the leading and lagging strands
DNA Pol α
what happens to DNA Pol α once a primer is put in place?
undergoes polymerase switching, whereby Pol α dissociates from the template and is replaced by Pol δ or ε
eukaryotic DNA pol(s) with high processivity; extend the primers on opposite strands of DNA; exhibit 3’ to 5’ exonuclease activity, thus having the potential to proofread
Pol δ (leading strand) and Pol ε (lagging strand)
what are the stages of DNA replication?
(1) initiation
(2) elongation
(3) termination
in initiation, a protein responsible for initiating replication binds to the origin of replication, which destabilizes and opens up double-stranded DNA, exposing ssDNA regions. what is this protein called?
DnaA
DnaA binds to a region of 9mers (found in oriC). this complex then undergoes a slight conformational change and associates with the region of 13mers, causing destabilization
site of replication initiation and separation of the double-stranded DNA
origin of replication
recruits the holoenzyme to bind to the newly formed replication fork and formally initiates replication; unwinds the dsDNA
helicase : direction of unwinding is 5’ to 3’
in the pdf : dnaA does the recruiting. in the book, helicase
what is needed for helicase to do its job of unwinding the strands?
ATP; achieved through hydrolysis
prevents single-stranded DNA from annealing
single-stranded binding proteins (SSBs); hairpin structures may be formed without SSBs
as unwinding proceeds, a coiling tension may be created ahead of the replication fork which produces ____.
supercoiling
supercoiling may be relaxed by a member of the larger group of enzymes called DNA topoisomerases which makes either single or double-stranded “cuts” and also catalyzes localized movements that have the effect of “undoing” the twists and knots created during supercoiling
DNA gyrase
these various reactions (undoing twists and releasing strands after) are driven by the energy released during ATP hydrolysis
a short segment of RNA primer which is complementary to DNA is synthesized to achieve a free hydroxyl group. this synthesis is directed by a form of RNA polymerase called ____.
all synthesis follows the 5’ to 3’ direction
primase
in elongation, this type of DNA pol synthesizes the leading strand continuously in the 5’ to 3’ direction
DNA Polymerase III
DNA synthesis is initiated at specific sites along each template strand
as the fork progresses, many points of initiation are necessary on the opposite DNA template (since DNA pol synthesizes in 5’ to 3’), resulting in a strand called ___.
lagging strand
enzymes that excises or removes RNA primers and fills in gaps by replacing missing nucleotides
DNA Polymerase I
enzyme that seals the nicks in the DNA/join the fragments together by catalyzing the formation of phosphodiester bonds
DNA ligase
termination begins when the replication fork collides at the ____.
terminus of replication
terminus of replication : opposite side of the chromosome from the origin
terminus of replication contains specific DNA sequences called ___.
Ter sites
proteins that recognize ter sites and block progress of the replication forks
tus proteins
once the replication fork collides at the terminus of replication, tus proteins bind with ter sites. this tus-ter complex then creates a physical block, which hinders the progress of replication fork and ensures correct termination
facilitates DNA partitioning in daughter cells during cell division
FtsZ
how does proofreading work during DNA replication?
(1) proofreading begins at time of nucleotide insertion
(2) mismatched nucleotide is excised from growing DNA strand
(3) correct nucleotide is inserted into growing DNA strand
proofreading increases the fidelity of synthesis by a factor of 100
replication through chromatin in eukaryotes
(1) removal and modification of nucleosomes and other DNA-binding proteins
(2) tight coupling of DNA synthesis and histone synthesis during S phase
(3) rapid reassociation of histones and non-histone proteins after replication
(4) assembly of new nucleosomes behind the replication fork through chromatin assembly factors (CAFs)
problems associated with linear DNA ends of eukaryotes
(1) double stranded “ends” of DNA molecules at the termini of linear chromsomes potentially resembles the double-stranded breaks (DSBs) that can occur when a chromosome becomes fragmented internally as a result of DNA damage.
🔸this may be targeted by DNA repair mechanisms
🔸vulnerable to nuclease degradation
(2) during DNA replication, DNA polymerases cannot synthesize new DNA at the tips of single-stranded 5’ ends
how are the replication problems at eukaryotic chromosome ends dealt with?
addition of nucleotides on telomeres via telomerase
region of repetitive DNA sequences at the end of a chromosome. which strand of the DNA is G-rich?
telomeres; 3’ strand
a ribonucleoprotein enzyme capable of adding several more repeats of the nucleotide sequence to the 3’ end of the G-rich strand
telomerase
serves as (1) a “guide” to proper attachment of the enzyme to the telomere and (2) a “template” for synthesis of its DNA complement
telomerase RNA components (TERC)
catalytic subunit of the telomerase enzyme
telomerase reverse transcriptase (TERC)
