Enzymes

Enzymes

1. DNA replication

   • 3. DNA polymerase
     • Catalyses the assembly of nucleotides into new DNA strands.
   • 1. Helicases
     • Separate base pairs, so separate the two DNA strands
   • 2. topoisomerase
     • Nicks the overwound strand of DNA at the replication fork and then reseals it after it has unwound.
   • 6. nucleases
     • Breaks links between nucleotides
   • 5. DNA ligase
     • Follows along removing RNA primers, filling in nucleotide gaps and ligating the fragments together to form a single DNA strand.
   • 4. primase
     • Generates an RNA primer that initiates the beginning of a new Okazaki fragment on the lagging strand

2. DNA repair

   • Mismatch repair
     • 1. MMR complex
       • Nicks the new strand close to length distortion (caused by different length of mismatched bases compared to correctly matched ones) and unwinds a short stretch of the helix.
     • 3. A DNA polymerase
       • A DNA polymerase then fills the gap left in the strand by synthesising new DNA in the 5' to 3' direction.
     • 2. exonuclease
       • Starting at the nick the exonuclease degrades a section of the DNA by removing one nucleotide at a time.
     • 4. DNA ligase
       • Finishes the job by joining (ligating) the sugar phosphate backbone of the newly synthesised section of DNA.
   • base excision repair
     • When DNA is damaged by a variety of agents such as UV or chemicals causing lesions. Proteins for repair have been highly conserved across species. e.g. deanimation of cytosine.
     • 1. DNA glycosylase
       • Catalyses the cleavage of a nucleotide from the sugar backbone. Several types for specificity for different bases. eg uracil DNA glycosylase. This leaves an abasic site
     • 2. nucleases
       • Nucleases remove the sugar-phosphate backbone of the abasic site.
     • 3. DNA polymerase
       • Adds the correct nucleotide.
     • 4. ligase
       • Ligase repairs the sugar phosphate backbone.
   • nucleotide excision repair
     • e.g. Repair of thymine dimers produced by covalent linking of thymine and cytosine due to UV-B (315 - 280 nm)
     • 1. Nucleases
       • Cleave the section of DNA with the distortion caused by the dimer.
     • 2. DNA helicase
       • Unwinds and separates the two strands, thus releasing the damaged section.
     • 3. DNA polymerase?
       • Book just says new DNA is synthesised.
     • 4. ligase
       • ligates the sugar phosphate backbone.
   • Double strand break repair
     • non-homologous end joining
     • homologous end joining

3. gene cloning

   • restriction endonucleases
     • Target specific DNA sequences and cleaves them. For example it will cleave and open the circle of a plasmid to be used for recombining with DNA fragment of interest in gene cloning.
   • DNA ligase
     • When incubated with DNA ligase the DNA of interest will recombine with the open circular plasmid DNA molecule to produce recombinant plasmids.

4. telomerase

   • Adds new repeat sequences to the ends of chromosomes, mostly on stem cells, germ cells or less usefully cancer cells.

5. reverse transcriptase

   • responsible for the synthesis of DNA from viral RNA in reverse transcription.

6. DNA dependent RNA polymerase

   • Used for synthesis of RNA via transcription.

7. bacterial lac operon

   • lacZ beta-galactosidase
     • breaks down lactose into galactose and glucose
   • lacY betagalactoside permease
     • a transport protein that transports lactose into the cell
   • lacA beta-galactosidase transacetyllase
     • function unclear

8. histone acetylases

   • Specific transcription factors activate promoters by changing the local structure of the heterochromatin at the promoter to improve the access to the DNA. This transcriional activator attracts enzymes called histone acetylases which catalyse the additioni of acetyl groups to lysine in the histone protein. Acetylation attraccts chromatin remodelling proteins whose function is to remove nucleosomes or slide them apart to expose the DNA.

9. guanylyl transferase

   • catalyses the capping of the 5' end during transcription

10. ribonucleases

    • Constantly attack mRNA and help to regulate RNA turnover, controlling the amount of mRNA in the cell. They remove nucleotides from the polyA tail at the 3' end of the mRNA. 200 long but eventually becomes too short.