Double strand break repair by protein repair machines

DNA synthesis
1. Requires
  1. dGTP, dATP, dCTP and dGTP
    1. 3 phosphoryl groups attached to 5' hydroxyl of 2'deoxyribose
      1. Inner most phosphoryl group: a phosphate
      2. Outer most phosphoryl groups: B and y phosphate
  2. Primer:template junction
    1. Primer is complementary to but shorter than template
      1. Primer exposed to 3'OH adjacent to ss region of template
      2. Only the primer is a substrate as it is chemically modified
    2. Template provides ssDNA that directs addition of nucleotides
      1. Provides info necessary to pick up when nucleotides are added
2. Extends 3' end of primer
  1. Phosphodiester bond formed in sn2 reaction
    1. 3' end of primer attacks a phosphoryl group of incoming nucleoside
      1. Pyrophosphate released from B and y phosphates
  2. Template strand directs which 4 nucleoside triphosphates are added
3. DNA polymerases
  1. Catalyse synthesis
  2. Monitors ability of incoming nucleotide to form a base pair
  3. When correct base pair is formed 3'OH of primer and a phosphate of incoming nucleoside in optimum position for catalysis
  4. Incorrect base pairing
    1. Lower rates of nucleotide addition
    2. Catalytically unfavourable
  5. Distinguish between rNTPs and dNTPs
    1. rNTPs are sterically excluded from DNA polymerase active site
      1. Nucleotide binding pocket is too small
        Space is occupied by 2 amino acids that make van der Waals contact with sugar ring
      2. Changing amino acids in pocket causes reduced discrimination
  6. Resembles a hand that grips template:primer junction
    1. DNA substrate sits in a large cleft that resembles a closed hand
    2. 3 domains
      1. Thumb
        Interacts with most recently synthesised DNA
        Maintains correct position of primer
        Maintains strong association between DNA polymerase and its substrate
      2. 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
      3. 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
1. Catalysis is rapid
2. Capable of adding 1000 nucleotides per second to primer strand
3. Speed of DNA synthesis due to processive nature of DNA polymerase
4. Degree of processivity defined by number of nucleotides per minute
5. Rate of DNA synthesis drastically increased by adding multiple nucleotides per binding event
6. Initial binding of polymerase to primer:template junction is rate limiting step
  1. Once bound, addition of nucleotide is very fast
7. Sequence independent nature of interactions permits easy movement of DNA
8. Each time a nulceotide is added, DNA partially releases from DNA
9. DNA rapidly rebinds to DNA as it is shifted 1 bp
10. Increase in processibity by binding of DNA polymerase and sliding clamp protein the completely encircles DNA
Tranlesion synthesis
1. Allows replication to proceed over DNA damage
2. DNA polymerase cannot replicate over a lesion
3. Highly error prone
4. E.coli
  1. UmuD is cleaved to shorter form UmuD'
    1. UmuD' forms a complex with UmuC
      1. Creates polymerase V which replicates past lesion
  2. Proteins from Y family of polymerases
    1. Independent of base pairing
      1. Enzyme is not reading sequence information
  3. DNA damage leads to proteolytic destruction of transcriptional repressor LexA
    1. Cleavage of LexA and UmuD stimulated by RecA
      1. RecA stimulated by ssDNA damage
5. Eukaryotes
  1. Triggered by chemical modification of sliding clamp PCNA
    1. PCNA anchors replicative polymerase to DNA template
      1. 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
  2. DNA polymerase n promotes TLS

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Double strand break repair by protein repair machines

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	Criado por sophie_connor mais de 11 anos atrás