110092
Description
Mind Map by sophie_connor, updated more than 1 year ago
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Created by sophie_connor
almost 11 years ago
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Double strand break repair
by protein repair machines
- DNA synthesis
- Requires
- dGTP,
dATP,
dCTP and
dGTP
- 3 phosphoryl groups attached to 5'
hydroxyl of 2'deoxyribose
- Inner most phosphoryl
group: a phosphate
- Outer most phosphoryl
groups: B and y
phosphate
- Inner most phosphoryl
group: a phosphate
- 3 phosphoryl groups attached to 5'
hydroxyl of 2'deoxyribose
- Primer:template
junction
- Primer is
complementary to
but shorter than
template
- Primer exposed to
3'OH adjacent to ss
region of template
- Only the primer is a
substrate as it is
chemically modified
- Primer exposed to
3'OH adjacent to ss
region of template
- Template provides
ssDNA that directs
addition of
nucleotides
- Provides info necessary to pick
up when nucleotides are added
- Provides info necessary to pick
up when nucleotides are added
- Primer is
complementary to
but shorter than
template
- dGTP,
dATP,
dCTP and
dGTP
- Extends 3' end of primer
- Phosphodiester bond
formed in sn2 reaction
- 3' end of primer attacks a
phosphoryl group of incoming
nucleoside
- Pyrophosphate released
from B and y phosphates
- Pyrophosphate released
from B and y phosphates
- 3' end of primer attacks a
phosphoryl group of incoming
nucleoside
- Template strand
directs which 4
nucleoside
triphosphates are added
- Phosphodiester bond
formed in sn2 reaction
- DNA polymerases
- Catalyse
synthesis
- Monitors ability of incoming
nucleotide to form a base pair
- When correct base pair is formed 3'OH of
primer and a phosphate of incoming
nucleoside in optimum position for catalysis
- Incorrect base
pairing
- Lower rates of
nucleotide
addition
- Catalytically
unfavourable
- Lower rates of
nucleotide
addition
- Distinguish between
rNTPs and dNTPs
- rNTPs are sterically
excluded from DNA
polymerase active site
- Nucleotide binding
pocket is too small
- Space is occupied by 2 amino
acids that make van der Waals
contact with sugar ring
- Space is occupied by 2 amino
acids that make van der Waals
contact with sugar ring
- Changing amino acids
in pocket causes
reduced discrimination
- Nucleotide binding
pocket is too small
- rNTPs are sterically
excluded from DNA
polymerase active site
- Resembles a hand that grips
template:primer junction
- DNA substrate sits in a
large cleft that resembles
a closed hand
- 3 domains
- Thumb
- Interacts with most
recently synthesised
DNA
- Maintains
correct position
of primer
- Maintains strong
association between DNA
polymerase and its
substrate
- Interacts with most
recently synthesised
DNA
- Palm
- B sheet
- Primary elements
of catalytic site
- DNA polymerase binds 2
divalent metal ions
- Alter chemical environment
around base pair and 3'OH of
primer
- One metal ion reduces affinity of
3'OH for the primer
- Generates 3'O- primed for
nucleophilic attack of the a
phosphate of the incoming
dNTP
- Second metal ion coordinates
negative charges of B and y
phsophates of the dNTP and
stabilises pyrophosphate
- Second metal ion coordinates
negative charges of B and y
phsophates of the dNTP and
stabilises pyrophosphate
- Generates 3'O- primed for
nucleophilic attack of the a
phosphate of the incoming
dNTP
- One metal ion reduces affinity of
3'OH for the primer
- Alter chemical environment
around base pair and 3'OH of
primer
- Monitors base
pairing of
recently added
nucleotides
- Makes extensive
hydrogen bond
contacts with base
pairs
- Mismatched DNA
interferes with minor
groove contacts and slows
catalysis
- Slowed catalysis and
reduced affinity allows
release of primer strand
- Strand binds proofreading
nuclease and removes
mismatched DNA
- Strand binds proofreading
nuclease and removes
mismatched DNA
- Slowed catalysis and
reduced affinity allows
release of primer strand
- B sheet
- Finger
- Important
for catalysis
- Several residues
bind incoming
dNTP
- A correct base pair
formed between dNTP
and template
- Finger domain moves to
enclose dNTP
- Closed form stimulates catalysis
- Moves incoming nucleotide in
close contact with catalytic metal
ions
- Moves incoming nucleotide in
close contact with catalytic metal
ions
- Closed form stimulates catalysis
- Finger domain moves to
enclose dNTP
- Associates with
template region
- Leading to turn of
phosphodiester
backbone
- Bend exposes first
template base
- Avoids confusion
concerning which template
base should pair next
- Avoids confusion
concerning which template
base should pair next
- Bend exposes first
template base
- Leading to turn of
phosphodiester
backbone
- Important
for catalysis
- Thumb
- DNA substrate sits in a
large cleft that resembles
a closed hand
- Catalyse
synthesis
- Requires
- DNA polymerases are
processive enzymes
- Catalysis is rapid
- Capable of adding 1000
nucleotides per second to
primer strand
- Speed of DNA synthesis
due to processive nature of
DNA polymerase
- Degree of processivity defined
by number of nucleotides per
minute
- Rate of DNA synthesis drastically
increased by adding multiple
nucleotides per binding event
- Initial binding of
polymerase to
primer:template junction is
rate limiting step
- Once bound, addition
of nucleotide is very fast
- Once bound, addition
of nucleotide is very fast
- Sequence independent
nature of interactions
permits easy movement of
DNA
- Each time a
nulceotide is
added, DNA
partially releases
from DNA
- DNA rapidly rebinds to
DNA as it is shifted 1
bp
- Increase in processibity by
binding of DNA polymerase
and sliding clamp protein the
completely encircles DNA
- Catalysis is rapid
- Tranlesion synthesis
- Allows replication
to proceed over
DNA damage
- DNA polymerase cannot
replicate over a lesion
- Highly error prone
- E.coli
- UmuD is cleaved to shorter form UmuD'
- UmuD' forms a complex with UmuC
- Creates polymerase V which replicates
past lesion
- Creates polymerase V which replicates
past lesion
- UmuD' forms a complex with UmuC
- Proteins from Y family
of polymerases
- Independent of base pairing
- Enzyme is not
reading sequence
information
- Enzyme is not
reading sequence
information
- Independent of base pairing
- DNA damage leads to
proteolytic destruction of
transcriptional repressor
LexA
- Cleavage of LexA and
UmuD stimulated by RecA
- RecA stimulated by
ssDNA damage
- RecA stimulated by
ssDNA damage
- Cleavage of LexA and
UmuD stimulated by RecA
- UmuD is cleaved to shorter form UmuD'
- Eukaryotes
- Triggered by chemical
modification of sliding
clamp PCNA
- PCNA anchors replicative
polymerase to DNA
template
- Ubiquitination of PCNA
triggers TLS
- Clamp recruits translesion
polymerase which contains
domains that recognise and bind
to ubiqutin
- Translesion polymerase displaces
replicative polymerase which can
bind ubiquitin
- Translesion polymerase displaces
replicative polymerase which can
bind ubiquitin
- Clamp recruits translesion
polymerase which contains
domains that recognise and bind
to ubiqutin
- Ubiquitination of PCNA
triggers TLS
- PCNA anchors replicative
polymerase to DNA
template
- DNA polymerase n promotes TLS
- Triggered by chemical
modification of sliding
clamp PCNA
- Allows replication
to proceed over
DNA damage
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