Chapter 6: DNA Replication Flashcards
because replication is (), each strand of the parental double helix acts as a template for the synthesis of a new daughter DNA strand
semi-conservative
3 phases of DNA synthesis
- initiation
- elongation
- termination
replication starts at special sites called ()
origins of replication
replication moves away from the origin in (1), forming a (2)
- both directions
- replication bubble
the DNA double helix is opened at the origin of replication and unwound to form ()
replication forks
at replication forks, () is exposed and DNA synthesis can occur
single-stranded DNA (ssDNA)
for bacteria, the origin of replication is typically a single site called ()
ori
in most eukaryotes, the origins of replication are () along the chromosome
multiple, not sequence-specific
the origin of replication is recognized by an () that opens up the double helix and recruits helicases
initiator protein
() unwind the double helix to expose ssDNA
DNA helicases
ssDNA is coated with () to prevent it from forming secondary structures or re-pairing, and to protect it from endonucleases
ssDNA binding proteins
DNA synthesis is carried out by (), but it can only add nucleotides to an existing 3’ end
DNA polymerase (III)
because DNA polymerase cannot synthesize new strands de novo, DNA synthesis needs a (1) synthesized by an RNA polymerase called (2)
- primer
- primase
DNA polymerase us recruited by the (1) to the DNA at the (2)
- sliding clamp
- primer 3’ terminus
after the RNA primer is synthesized, the (1) loads the (2) onto the DNA template strand
- clamp loader
- sliding clamp
in eukaryotes, the first few nucleotides are synthesized by (1), which forms (2)
- DNA polymerase alpha
- polymerase alpha - primase complex
DNA synthesis on both template strands occurs in a () direction
5’ to 3’
due to the 5’ to 3’ direction of DNA synthesis, synthesis is continuous on the (1), but discontinuous on the (2)
- leading strand
- lagging strand
polymerases, helicases, and primases are organized into the () at the replication fork
replisome
(dimeric) polymerases on both leading and lagging strands travel () at the replication fork
together behind the helicases
termination of DNA synthesis occurs when:
- 2 different forks meet
- the fork reaches the end of a linear chromosome
- polymerase meets the previously replicated strand
because RNA primers were used to start DNA synthesis, they must be () during termination
removed and replaced with DNA
in the case of discontinuous synthesis or replacement of RNA primers, () connects the adjacent strands
DNA ligase
bacterial DNA synthesis termination occurs at a specific region called ()
ter
DNA polymerases catalyze the (1) to the (2) in the newly growing strand
- addition of a new nucleotide
- 3’ OH of the last nucleotide
the structure of DNA polymerase resembles a right hand, with 3 domains called:
- thumb
- palm
- fingers
ssDNA is fed past the () domain of DNA polymerases
fingers
nucleotide addition is catalyzed in the active site in the () domain of DNA polymerase, which forms a cleft into which the growing dsDNA fits
palm
the () domain of DNA polymerase holds the elongating dsDNA
thumb
DNA polymerase adds nucleotides (dNTPs) to the nascent DNA strand by ()
nucleophilic attack
the kind of nucleotides added by DNA polymerase to the growing DNA strand
deoxyribonucelotide triphosphates (dNTPs)
the active site of DNA polymerase catalyzes a (1) reaction linking the 5’ phosphate of the incoming dNTP to the 3’ OH of the growing DNA to form a (2)
- phosphoryl transfer
- phosphodiester bond
nucleophilic attack by the nascent chain 3’ OH on the alpha-phosphate of the incoming dNTP releases ()
pyrophosphate (PPi)
during the phosphoryl transfer reaction, DNA polymerase uses () in the active site to promote the reaction
2 metal ions (Mg2+)
subsequent () drives the phosphoryl transfer reaction in DNA synthesis forward
hydrolysis of released pyrophosphate
initiator proteins bind to () regions on the unwound DNA strands to allow helicases to continue unwinding the DNA
A-T
why are A-T rich regions easier to unwind than G-C rich areas
A is bound to T by only 2 H-bonds, as compared to the 3 H-bonds connecting G and C
the A-T rich region is called a ()
DNA unwinding element
for bacteria and a few eukaryotic viruses, the DNA unwinding element consists of ()
specific DNA sequence elements
in most eukaryotes, the DNA unwinding element lacks defined sequence elements; instead, the initiator proteins bind at ()
many sites
initiator proteins are (), which are associated with a variety of cellular activities
AAA+ ATPases
in bacteria, the initiator protein is called ()
DnaA
in eukaryotes, the initiator protein is called the ()
origin recognition complex (ORC)
the ORC is composed of (1) subunits, labeled (2)
- 6
- Orc1-6
a special case for eukaryotes: in (), ORC binds to a specific DNA sequence
S. cerevisiae
the ORC in S. cerevisiae binds to a specific DNA sequence called the ()
ARS (autonomously replicating sequence)
the binding of ORC to DNA is likely influenced by the ff.
- chromatin structure
- nucleosomes
- sequence-specific DNA binding proteins
origins of replication are activated at random throughout the ()
S phase
in E. coli, the origin (called 1) has a 245 bp sequence, with seven 9bp (2) that bind DnaA
- OriC
- DnaA boxes
when bound to ATP, the AAA+ domains of DnaA multimerize into a ()
spiral filament
the () between the E. coli DNA and initiator proteins at the origin distorts the DNA, which facilitates unwinding at the adjacent AT-rich region
filament interaction
as well as generating ssDNA, DnaA also recruits bacterial () to the origin
bacterial DNA helicase DnaB
in E. coli, helicase DnaB is loaded onto the DNA by ()
helicase loader DnaC
bacterial helicase is loaded onto (1), while eukaryotic helicase is loaded onto (2)
- ssDNA
- dsDNA
the ORC recruits 2 proteins:
- Cdc6
- Cdt1
the Cdc6 and Cdt1 proteins recruited by the ORC sequentially load 2 ring-shaped () in a head-to-head orientation
MCM2-7 hexamers
the MCM2-7 pair loaded by the Cdc6 and Cdt1 proteins is then activated by accessory proteins such as ()
Cdc45 and Sld3
other proteins are loaded (including polymerase) to the activated MCM2-7 pair, forming the (1), which can be activated by (2) to unwind DNA and start replication
- full helicase complex
- phosphorylation
the full helicase complex is called the (1) for (2)
- CMG complex
- Cdc45, MCM, GINS
the eukaryotic replicative helicase CMG is a ring composed of ()
6 MCM subunits, plus additional factors
the ssDNA binding protein for bacteria is called ()
SSB protein
the ssDNA binding for eukaryotes is called ()
replication protein A (RPA)
the primer in bacterial DNA synthesis is a ()
short piece of RNA
on the lagging strand, the discontinuous synthesis of DNA results in the formation of ()
Okazaki fragments
the primer in eukaryotic DNA synthesis is a ()
short piece of RNA+DNA
the DNA polymerase in the polymerase alpha-primase complex is not replicative polymerase, so to complete (1), it must add (2) before to the primer allowing replicative polymerases to take over
- polymerase switching
- a short piece of DNA
addition of (1) after primer synthesis is done by the interaction with the sliding clamp, which is loaded at the (2) by the clamp loader
- replicative DNA polymerase
- template-primer junction
in bacteria, the sliding clamp is called
beta protein
in eukaryotes, the sliding clamp is called ()
PCNA
the high processivity of replicative DNA polymerase is due to the ()
sliding clamp that keeps it tethered to the DNA
the clamp loader (called 1 in eukaryotes) is a (2) ring structure, and some clamp-loader subunits are (3)
- replication factor C, RFC
- 5-subunits
- AAA+ ATPases
the cycle of sliding clamp loading to DNA is driven by (1) driven about by the binding of (2) and its subsequent hydrolysis
- conformational changes
- ATP
in the absence of ATP, the clamp loader has () for the sliding clamp
low affinity
the clamp loader-sliding clamp complex has a high affinity with the ()
primer-template junction
binding of the clamp loader-sliding clamp complex stimulates (), which closes the sliding clamp and releases the clamp loader
ATPase activity
after the release of the clamp loader, the sliding clamp remains associated with DNA, and recruits DNA polymerase to the primer-template junction via ()
short polymerase binding motifs
the clamp loader must be () in order to repeat the cycle
recharged with ATP
polymerase switching in eukaryotes is needed because polymerase alpha is already at the primer 3’ end and must be replaced with replicative polymerase (1) on the lagging strand or (2) on the leading strand
- delta
- epsilon
polymerases can be grouped according to the () of the rest of the protein
evolutionary lineage
polymerases are more similar within groups than within organisms, suggesting ()
early divergence of polymerase groups
DNA polymerases that copy RNA into DNA
reverse transcriptases
reverse transcriptases are typically encoded by viruses and by DNA elements in eukaryotes called ()
retrotransposons
a specialized reverse transcriptase that is needed to synthesize chromosome ends
telomerase
3 quality control steps of DNA replication
- low polymerase error rate
- proofreading by the polymerase
- mismatch repair
during addition, the polymerase recognizes the correct nucleotide due to the () when base-paired with the template strand
precise fit in the active site
proofreading by the replicative polymerase is achieved by (), where an incorrect nucleotide is removed before the next nucleotide is added
3’ to 5’ exonuclease activity
in the event of the addition of an incorrect nucleotide to the newly-synthesized strand, it is moved to the () and removed
exonuclease site
replicative polymerases stay attached for many 1000s of nucleotides; this synthesis is ()
processive
in (), Okazaki fragments are joined after synthesis; this process is different in eukaryotes and bacteria
Okazaki fragment maturation
exonucleases can only cut from
a strand end
in E. coli, () disassociates from the DNA when it reaches the next primer
DNA polymerase III
in E. coli, () removes the RNA primer and then fills the gap with DNA; however, it leaves a nick in the DNA because it cannot attach the end of the synthesized DNA to the next DNA fragment
DNA polymerase I
() seals the DNA nick in E. coli
DNA ligase
DNA polymerase I in E. coli is able to remove the RNA primer due to () activity
5’ to 3’ exonuclease
in eukaryotes. () continues to make DNA when it meets the RNA primer
DNA polymerase delta
the flap created by DNA polymerase delta in eukaryotic Okazaki fragment maturation is cleaved by ()
Flap endonuclease 1 (Fen1)
Endonucleases can cut and digest DNA strands from the ()
middle
in eukaryotes, if Fen1 doesn’t cleave the DNA, a long flap forms, which is then cleaved by ()
Dna2
the bacterial replisome has 2 copies of DNA polymerase III, linked by (), which associates with the clamp loader and helicase
tau
leading and lagging strand synthesis in eukaryotes is coordinated at the replication fork by ()
Ctf4
Ctf4 in eukaryotes couples (1), (2) and (3) at the fork
- helicase CMG
- polymerase epsilon
- polymerase alpha-primase
there is no eukaryotic equivalent of bacterial tau protein; instead, eukaryotic DNA strands are coupled through ()
Ctf4-polymerase alpha-primase hub
unwinding of DNA introduces torsional stress, resulting in (1) ahead of the fork = (2)
- overwinding
- positive supercoiling
substrate of a topoisomerase and the product are chemically identical, but ()
topologically different
replication of bacterial circular chromosomes results in daughter molecules that are still interlinked - ()
catenated
bacterial circular chromosomes are unlinked (decatenated) by ()
topoisomerases (type1A or type II)
() topoisomerases break 1 DNA strand and do not require ATP
type I
type I topoisomerases have an active site () that attacks the phosphodiester bond of one strand in duplex DNA
tyrosine-OH
the attacking of topoisomerase I results in a transient covalent phosphodiester linkage between ()
tyrosine and 5’ or 3’ end of cleaved strand
type IA topoisomerases use the () mechanism to rebind cut strands
strand passage
how to type IB topoisomerases rebind the cut strands
allow the free end to rotate to release supercoils
() topoisomerases are dimeric and break both DNA strands and do require ATP
type II
type II topoisomerases use the () mechanism to rebind cut strands
strand passage
an unusual type II topoisomerase that introduces negative supercoils -> makes strand unwinding ahead of fork more favorable
bacterial DNA gyrase
ter sites in bacterial DNA are bound by the (), which is orientation specific and allows passage in only 1 direction
Tus proteins
bacterial replication forks meet in a termination zone that has ()
10 ter sites
initiation of replication in bacteria is tightly regulated by factors such as (1) and (2)
- cell size
- nutritional state
primary methods of regulating initiation of replication in E. coli
- regulatory inactivation of DnaA (RIDA)
- initiation at oriC by methylation
- reduction of available DnaA amount by sequestration
- recycling DnaA-ADP to DnaA-ATP
after initiation, the () protein stimulates hydrolysis of DnaA-ATP to DnaA-ADP; used in RIDA
Hda
main consequence of RIDA
DnaA-ADP dissociated from oriC and cannot reinitiate firing -> prevents overstimulation of replication initiation
major regulatory step of preventing initiation in E. coli
regulatory inactivation of DnaA
enzyme that methylates the A residue in the sequence GATC
DNA adenine methylase (Dam)
action of Dam results in () of DNA, where only 1 of the 2 strands has a methyl tag
hemi-methylation
protein that binds to hemi-methylated GATC sites
SeqA
main consequence of SeqA binding to GATC sites
SeqA binding temporarily blocks binding of methylase and thus prevents complete methylation of DNA -> prevents DnaA binding for replication initiation temporarily
DNA region in E. coli that lies close to oriC and where (about 25% of) DnaA can also bind
datA DNA region
DNA regions far away from oriC; serve as cofactors to stimulate the exchange of ADP to ATP on DnaA
DARS regions (DnaA reactivating sequences)
main difference between bacterial initiation and eukaryotic initiation (of replication)
- origins of bacteria can fire initiation more than once in the cell cycle
- replication origins in eukaryotes must only fire a maximum of only once per cell cycle
initiation at each oriC in a bacterial cell is (1) - therefore the number of oriC in a cell is always a power of (2)
- synchronous
- two
selection of origins in G1 of eukaryotic cell cycle
origin licensing
in the (1), eukaryotic helicases are activated to initiate origin unwinding; once fired, origins cannot be reused until next (2)
- S phase
- G1 phase
() are proteins involved in restricting initiation steps and origin firing to the appropriate timing in eukaryotes
cell cycle kinases
cell cycle kinases mainly work by () target proteins
phosphorylating
how do yeast cells prevent helicase loading outside of G1 phase
regulation is achieved by exporting proteins from the nucleus
how do metazoa prevent helicase loading outside of G1 phase
- proteolysis of replication complex proteins after S-phase
- binding of Geminin to Cdt1
how does Geminin binding to Cdt1 regulate initiation in metazoa
when bound to Geminin, Cdt1 cannot load MCM to origin of replication
related factors that control whether or not a pre-replication complex will fire in the cell cycle
- origin efficiency
- origin timing
probability that an origin will fire
origin efficiency
timing (earlier or later) of firing compared to other origins
origin timing
most origin firing in eukaryotes is thought to be largely stochastic, with evidence of () as a limiting factor
replication protein availability
what is the end-replication problem
mechanism for lagging strand synthesis cannot replicate the very end of a linear chromosome
consequence of the end-replication problem
substantial portions of chromosome ends could be lost over several rounds of replication
some causes of chromosome portion loss due to end-replication problem
- RNA primer removal
- dissolution of the replication fork
() leaves a gap which cannot be filled in at the end of linear chromosomes
RNA primer removal
many eukaryotes use () to circumvent the end-replication problem
telomeres
in () several copies of the linear chromosome are stuck together before replication, reducing the number of ends -> way to avoid end-replication problem
T4 viruses
in (), the two DNA strands are joined at the end, making a hairpin so replication can just continue around the loop -> way to avoid end-replication problem
vaccinia viruses
in (), replication is primed from a terminal protein -> way to avoid end-replication problem
bacteriophage phi29
telomeres are (); their ends get shorter after each round of replication
simple sequence repeats
telomeres can be elongated by adding () at the ends
new specific telomere sequences
in Drosophila, () transpose onto chromosome ends to maintain length
transposable elements
telomere length in most eukaryotes in maintained by (); they add telomere DNA sequences to the chromosome ends
telomerase
telomerase synthesizes one strand of the telomere - the (1) strand - at the (2) end
- G-rich
- 3’
if telomerase synthesizes the G-rich strand, the complementary C-rich strand is filled in by ()
DNA polymerase alpha
the action of telomerase results in (), which counterbalances loss due to incomplete end-replication
net elongation of telomere sequence
telomerase is a unique polymerase made of a catalytic protein (1) and an (2)
- reverse transcriptase
- integral bound RNA molecule
the catalytic protein in telomerase is specifically called (); it is well-conserved in eukaryotes
telomerase reverse transcriptase (TERT)
role of integral bound RNA molecule in telomerase
- provides template for synthesis of telomere repeats
- involved in enzyme activity
it is not yet understood how the distribution of telomere length in a cell is regulated, but () are thought to be involved
telomere binding proteins
2 factors that help establish equilibrium of telomere length
- number of telomeres elongated in a cell cycle
- number of repeats added in a single binding event
mutations in telomere binding proteins can lead to ()
aberrant telomere lengthening
short telomeres induces the () response
DNA damage
loss of telomerase or other factors can lead to ()
progressive telomere shortening
mutations in telomerase cause inherited degenerative diseases called ()
telomere syndromes
telomerase is the main way that cells maintain telomeres, but some cells can used specialized recombination called ()
gene conversion
gene conversion takes advantage of the () at telomere ends
repetitive sequences
in gene conversion, the 3’ end of one chromosome can () and provide a template for telomere elongation by DNA polymerase
invade another chromosome
chromatin consists of DNA plus specific proteins called (), which are bound to DNA
histone proteins
correct recruitment of histones and maintenance of chemical modifications (e.g. methylation) form the basis for ()
epigenetic inheritance
a process where the parental H3 and H4 histones are divided among the daughter cell (each get 1/2) -> allows histone modification state to be inherited by both cells
parental histone segregation
after parental histone segregation, new () are deposited and modified in line with the old histones
H3 and H4 histones
copying chemical “decorations” of the parent strand is important for eukaryotes because these help ()
dictate which genes to express in certain cells
re-assemble histones on new daughter strands after replication fork disrupts nucleosomes
histone chaperone proteins
proteins that help to disrupt the nucleosome ahead of the fork and transfer the old H3/H4 from ahead of the fork to behind the fork
- FACT (histone chaperone)
- ASF protein
as daughter strands are synthesized, new () histones are loaded to make half a nucleosome
H2A and H2B
new H3/H4 histones are assembled by () and then added to the initial half of replicated nucleosome (composed of H2A/B histones)
CAF1 protein
because synthesis of the main histone subunits is tightly coupled to DNA synthesis, they are called (1) or (2)
- replication-dependent histones
- S phase histones
newly synthesized H3 and H4 histones are acetylated at specific (1, AA) in their (2) tails
- lysine
- N-terminal
deposition of new acetylated H3/4 histones on newly replicated DNA is performed by (1, 2 proteins) plus other histone chaperones
CAF1 and ASF1
() binds strongly to H3/H4 and to the sliding clamp to target H3/H4 to DNA behind the fork
CAF1
() binds half a nucleosome tetramer (2 H3 and 2 H4) and deposits this on DNA with the help of CAF1
ASF1
