Ch 11 Lecture (DNA Replication) Flashcards
initiation
recognition of the origin of replication by the replicon
elongation
replication of the parental duplex by the replisome
replisome
assembles at an origin of replication
elongates
joining/termination
completion of replication process, includes separation or joining of daughter duplexes
DNA polymerases synthesize DNA in
semiconservative replication and DNA repair
All DNA polymerases synthesize
5’ to 3’
some repair polymerases function as
independent enzymes
replicases are incrporated with other enzymes into a large complex called the
replisome
E. coli polymerases
Polymerase III is the primary replicase
Polymerase II is required to restart the replication fork after it is stopped by damage
Polymerase I is involved in both error repair and replication
Polymerases IV and V are error-prone polymerases that allow bypass around DNA damage
replicases usually have nuclease activity, meaning they have a
3’ to 5’ proofreading function
DNA polmerase I
First DNA polymerase to be characterized
Two parts
Klenow fragment
Polymerase and 3’5’ proofreading function
Small fragment
5’3’ exonuclease function
Excises 10 bases at a time
Free base pairing between incoming dNTP and parental strand would allow for
mismatches
how do mismatches happen?
Twisting of the helix allows for nonstandard pairs such as C=T
Unique four nucleotide combinations called tautomers also allows nonstandard pairs such as C=A
High-fidelity DNA polymerases have a precisely constrained … that favors binding of standard base pairs
active site
Rate of incorporation of incorrect nucleotides is …X slower than correct nucleotides
10,000
DNA polymerases can also differentiate between
rNTPs and dNTPs
A sugar with a 2’-OH cannot be easily accommodated in
the nucleotide-binding pocket
processivity
The ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle
Frameshift fidelity is increased by enzyme processivity, especially in homopolymeric regions
many DNA polymerases have a structure that resembles a
right hand
palm
primary elements of the polymerase catalytic site
fingers
binds to incoming dNTPs and moves the correct dNTP into close contact with the polymerase catalytic site
thumb
Maintains the correct position of the 3’-OH and also a strong association between the polymerase and the parental strand to facilitate processivity
metal cations in the palm domain
One metal ion reduces the affinity of 3’-OH for its hydrogen
More nucleophilic
Other metal ion stabilizes the negative charges of the β- and gamma-phosphates of the incoming dNTP and the departing pyrophosphate
the palm domain is composed of
a β sheet and contains the primary elements of the catalytic site
Also contains two divalent metal cations that interact with the 3’-OH of the primer and the correct paired dNTP
If a newly added nucleotide is mismatched
Disruption of nonspecific hydrogen bonding between palm and base pairs in minor groove of new duplex leads to reduced catalysis rate
Fingers cannot rotate towards palm to bind and direct dNTP to active site
The unpaired 3’ region will move into the exonuclease domain
The DNA polymerase complex advances continuously when it synthesizes the
leading strand
The lagging strand is synthesized by
making short Okazaki fragments that are subsequently joined together
semidiscontinuous replication
The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously
Two functions are needed to convert duplex DNA to a single stranded form
A helicase is needed to separate the strands of DNA using energy provided by the hydrolysis of ATP
A single-strand DNA binding protein is required to maintain the separated strands
Most helicases are
multimeric proteins that initially encircle DNA at a single stranded region next to a duplex region
most helicases have
both a double stranded conformation and a single stranded conformation
the movement of helicases results in
unwinding of the DNA
How many ATP are hydrolyzed for each base pair unwound by helicase
one
Single stranded binding protein in E. coli is a
monomer that binds single stranded DNA cooperatively
Once binding has began on a DNA molecule
it is rapidly extended along length of ssDNA
Single stranded binding proteins are also needed for
repair and recombination mechanisms
All DNA polymerases require a free … to initiate DNA synthesis
3’-OH
The free 3’-OH can be provided by
- RNA primer
- Nick in DNA
Recombination - Preformed RNA (tRNA)
Retroviruses - Priming protein that provides a nucleotide with a free 3’-OH
Ser, Thr, Tyr for some linear phages
Only one priming event is required on the
leading strand
Only one priming event is required for each
Okazaki fragment
DnaG primase
DnaG primase is a special RNA polymerase in E. coli
Used only for DNA replication
10 bp primer
Recognizes 3’-GTC-5’ on the parental strand and begins RNA synthesis with pppAG
Multiple core polymerase enzymes are required to
synthesize the leading and lagging strands
In E. coli the Active replisome is an
asymmetrical trimer with one core polymerase on leading and two on lagging strand
The replisome in E. coli consists of
DNA polymerase III holoenzyme, primase, and helicase
The holoenzyme contains … catalytic core polymerase subunits
three
One on leading strand
Two on lagging strand
Composition of DNA polymerase III holoenzyme
Three copies of the polymerase III catalytic core
Three copies of the linker protein tau
Three copies of the clamp, each containing
One copy of the clamp loader
polymerase III catalytic core contains
One alpha subunit for DNA polymerase activity
One epsilon subunit for 3’to5’ proofreading exonuclease
One theta subunit used to stimulate exonuclease activity when mispaired nucleotides are present
linker protein tau
joins the core enzymes to the sliding clamp loader
clamp contains
β2 ring that binds the DNA for processivity
clamp loader
gamma complex of seven proteins that load the clamp on the DNA
Assembly of DNA polymerase III
- The clamp loader hydrolyzes ATP to bind β subunits to a template-primer complex
- Binding of β to DNA changes its affinity for DNA polymerase
- A core DNA polymerase binds to a clamp
- tau trimer binds the core polymerase and stimulates the binding of two core-β2 complexes to form a trimer
- DNA polymerase III holoenzyme is formed
As DnaB helicase moves along the lagging strand and denatures the parental duplex,
SSB coat the single stranded DNA that is produced
The clamp loader hydrolyzes ATP to bind β subunits to a template-primer complex
Loader binds ATP
β2 clamp binds to loader
Loader-ATP will open clamp and load it onto a template-DNA complex
Binding of DNA stimulates hydrolysis of ATP to ADP
Loader-ADP is released from closed clamp-DNA complex
The core on the leading strand displaces the SSB and
continues synthesis
On the lagging strand, the single stranded DNA that is produced is coated with
SSB and looped
The size of the loop … as helicase continues to denature the parental duplex
increases
Simultaneously, one of the lagging strand core polymerases (Lagging Core 1) will be synthesizing the … Okazaki fragment
previous
At the fork, when the DnaG primase component finds the correct sequence, it will begin
primer synthesis
Following completion of primer synthesis, the clamp loader will
load a new clamp onto the newly primed lagging strand
The other lagging strand core (Lagging Core 2) will bind the clamp and begin synthesis of an Okazaki fragment Before
synthesis of the previous Okazaki fragment is complete
When Lagging Core 1 is finished synthesizing its Okazaki fragment,
it will be released from the clamp and disassociate from the lagging strand
Interactions between DnaB helicase and tau results in an
increase in helicase and core enzyme activity by 10X
DnaG primase is the only replisome component that is not
tightly associated with the fork
The only association between DnaG and the other fork components occurs via
weak interactions with DnaB
After primer synthesis, DnaG is
released back into solution
A stronger association between DnaG and DnaB would result in
more frequent periods of interaction between the components and more priming events creating shorter Okazaki fragments
A weaker association between DnaG and DnaB would result in
less frequent interactions and fewer priming events creating longer Okazaki fragments
DNA polymerase I
removes the primer and replaces it with DNA
Mammalian DNA polymerases do not have
5’ to 3’ exonuclease activity
Okazaki fragment primer removal
Synthesis of an Okazaki fragment will displace the primer of the previous fragment as a “flap”
Base of the flap is cleaved by FEN1
DNA polymerase will fill the gap where primer was located
FEN1 also important for preventing hairpins from forming in areas of repeated sequences
DNA ligase creates a
phosphodiester bond that fills “nicks” in the DNA backbone
Uses an AMP intermediate
DNA ligase in replication
Connects the terminal 3′-OH produced by DNA Polymerase I to the 5′-P of the first nucleotide previously added by DNA Polymerase III
As a replication fork moves along dsDNA, it must
unwind and denature the parental duplex
For every 10bp replicated
one complete turn of the parental duplex is unwound
Parental duplex ahead of the fork cannot …, so it will eventually become …
freely rotate
overwound
Excess positive supercoiling is released via the action of
DNA topoisomerase enzymes
DNA topoisomerase I
No external energy used to create single strand breaks
DNA topoisomerase II
Uses ATP hydrolysis to create double strand breaks
Bacterial DNA gyrase
Able to disentangle any two duplexes
A replication fork stalls when it arrives at
damaged DNA
Mispaired nucleotide
DNA break
what percent of bacterial forks encounter an error during a replication event
18-50%
common occurrence
The replication complex must be replaced by a specialized DNA polymerase for
lesion bypass
error-prone polymerase
Polymerase on lesion strand is removed and replaced with an error-prone polymerase
Damage is bypassed
High fidelity polymerase replaces error-prone polymerase
The two replication forks usually meet
halfway around the bacterial chromosome
If replication forks travel beyond their half of the chromosome,
ter sites will initiate disassociation of DnaB
Ter sites serve as a
directional “trap”
Eukaryotic replication fork composition
One DNA polymerase alpha
One DNA polymerase epsilon
One DNA polymerase delta
DNA polymerase alpha
initiates DNA synthesis
contains primase domains
binds to the initiation complex
Synthesizes primer plus approximately 20 nucleotides of DNA
DNA polymerase epsilon
elongation of the leading strand
DNA polymerase delta
elongation of the lagging strand
Leading and lagging strand polymerases act
independently of each other
Polymerase alpha is replaced by
polymerase epsilon/delta on the leading/lagging strands for remaining DNA synthesis
Polymerase epsilon is highly processive due to its interaction with
CMG helicase (Cdc45-GINS-Mcm)
RFC clamp loader
PCNA processivity clamp
The roles of RFC and PCNA are analogous to that of the
gamma clamp loader and β2 clamp in prokaryotes
On the leading strand
CMG helicase moves along the strand and denatures parental duplex
Attached polymerase epsilon will continuously replicate
On the lagging strand
Polymerase alpha will prime each Okazaki fragment
After polymerase switch, polymerase delta will complete fragment
Eukaryotic Okazaki fragment length of approximately 200 nucleotides
There must be some disruption of … during DNA replication
nucleosome structure
Disruption is confined to
the immediate vicinity of replication fork
Requires synthesis of enough histone proteins to package entire genome during S phase
20 repeated copies of major histone genes in vertebrates
Histone mRNA levels increase 50X in S phase due to
Increased transcription
Reduced degradation
After DNA replication is complete, major histone mRNAs are degraded within minutes
Disassembly and reassembly of nucleosomes at replication fork is directly linked to
replisome
Nucleosome assembly
H32 -H42 tetramer binds to DNA, followed by stepwise addition of H2A-H2B dimers
Replication-coupled pathway
Process is facilitated by histone chaperones
Chromatin remodeling complexes
Reverse process for disassembly
Nucleosome disassembly occurs at the replication fork
H32 -H42 tetramer is released from the duplex but still retained at the fork via direct and indirect associations with the CMG helicase
H2A-H2B dimers are fully released and diffuse away
Histone chaperones used in replication-coupled assembly
FACT
Histone chaperones used in replication-coupled assembly
CAF 1
NAP 1
CAF1
Associated with PCNA clamp
Assembles “old” and “new” H32-H42 onto daughter duplex
NAP 1
Associated with PCNA clamp
Assembles “old” and “new” H2A-H2B onto daughter duplex
FACT
Associated with replisome
Has flexible domains that bind to and help disassemble nucleosomes at fork
May also hand H32 -H42 tetramers to replisome components that pass them behind fork to CAF1
replication coupled assembly
- Replication fork displaces octamers
- Octamers disassociate into H32-H42 tetramers and H2A-H2B dimers
- “Old” H32-H42 tetramers are transferred randomly to one of the two daughter strands by FACT and NAP1
- “Old” H2A-H2B dimers are released into the soluble histone pool
- “New” H32-H42 tetramers are assembled onto other daughter strand by CAF1
- “Old” or “new” H2A-H2B dimer chosen from the soluble pool by NAP1
- The H2A-H2B dimer is placed onto daughter strands by NAP1
The specific modifications of these tetramers will result in
recruitment of specific enzymes that are able to propagate modifications onto the H2A-H2B members of the nucleosome
Also able to propagate modifications onto neighboring nucleosomes
Old H32-H42 tetramers (with their associated modifications) are passed to
the daughter duplexes
