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110092
Double strand break repair by protein repair machines
Description
Protein Form and Function Mind Map on Double strand break repair by protein repair machines, created by sophie_connor on 26/05/2013.
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protein form and function
protein form and function
Mind Map by
sophie_connor
, updated more than 1 year ago
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Created by
sophie_connor
over 11 years ago
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Resource summary
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
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
Template provides ssDNA that directs addition of nucleotides
Provides info necessary to pick up when nucleotides are added
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
Template strand directs which 4 nucleoside triphosphates are added
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
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
Changing amino acids in pocket causes reduced discrimination
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
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
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
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
Associates with template region
Leading to turn of phosphodiester backbone
Bend exposes first template base
Avoids confusion concerning which template base should pair next
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
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
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
Proteins from Y family of polymerases
Independent of base pairing
Enzyme is not reading sequence information
DNA damage leads to proteolytic destruction of transcriptional repressor LexA
Cleavage of LexA and UmuD stimulated by RecA
RecA stimulated by ssDNA damage
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
DNA polymerase n promotes TLS
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