DNA Replication

Anmerkungen:

• Requirements for DNA to be a genetic material A) Replication: DNA must be accurately copied B) Repair: DNA must have a repair mechanism to correct mistakes C) Expression: Info must be able to decoded and expressed at any time.

1. DNA polymerase

   Anmerkungen:

   • It has many functions. It has polymerisation and it adds nucleotides. Exonucleases 3' to 5' removes nucleotides as a proof reading mechanism (checks back) Exonucleases 5' to 3' removes nucleotides as a repair mechanism for nicks and more importantly removal of RNA primers.
   1. Types

      Anmerkungen:

      • As known from the previous bubble, they have exonuclease activity. In prokaryotic cells they have DNA polymerase III and DNA polymerase I where they have distinctly different target sites. Polymerase III elongates the chain by DNA synthesis while Polymerase I remove RNA primers and fill it up with DNA.
      1. DNA Polymerase III

         Anmerkungen:

         • It is a holoenzyme. It has a few components: Core enzyme which has 5' to 3' polymerase and 3' to 5' exonuclease catalytic activity Clamp loader manuever DNA polymerase III accordingly. Sliding clamp is like a loop to hold the complex to the DNA.
      2. DNA Polymerase I

         Anmerkungen:

         • It has a 5' to 3' exonuclease activity which DNA polymerase III doesnt have. And it also have 3' to 5' exonuclease domain too. The role is to remove RNA primers by "nick translation' This eventually need DNA ligase to seal up.
2. Template DNA strand
3. Primer with a free recessed 3'OH group

   Anmerkungen:

   • The primers are RNA primers THe 5' ends contains fragments of RNA complementary to the template DNA chain. One RNA is needed for leading while multiple for lagging strand. It will be replaced with DNA as it matures.
4. All 4 dNTP and Mg2+
5. Origin of Replication

   Anmerkungen:

   • A sequence of DNA where it indicate the DNA polymerase where to be cleaved. Bacteria cleave on one site.  1000 basepairs per seconds Eukaryotic DNA cleaves on multiple sites. 10 to 100 basepairs per second
   1. Origin

      Anmerkungen:

      • In E.Coli, the origin is consists of AT rich area and it separates into single strand by DnaA (helicase and gyrase)
6. Helicase

   Anmerkungen:

   • It unwinds the DNA strand by breaking the H-bonds.
7. DNA gyrase

   Anmerkungen:

   • DNA gyrase is a type II topoisomerase. It cleaves DNA in 2 locations to relieve supercoiling. Hence it is always a coil unwound from normal DNA
8. Single Strand DNA binding protein (SSB protein)

   Anmerkungen:

   • It coats the single stranded DNA to prevent it from being binded again to form dualstrand again.  This must be removed before replication can be proceeded.
9. Primase

   Anmerkungen:

   • In conjunction with the primase it forms the primosome. It goes forward and backwards to synthesize the complementary RNA primer for the lagging strand. In essence, the whole replication machinery is called the replisome. It has a special looping arrangement for the DNA's lagging strand to be synthesized.
10. DNA ligase

    Anmerkungen:

    • Join discontinuous strands of Okazaki fragments together.

Anmerkungen:

• Need DNA polymerase and a template for DNA replication for the DNA to work properly  Enzyme: DNA-directed DNA polymerase Template: Single stranded DNA because a double strand is highly associated with each other via hydrogen bonds. Mechanism: Nucleophillic attack on growing DNA chain. Driving force: Elimination of the Ppi.

1. Direction

   Anmerkungen:

   • Elongate at direction from 5' to 3'. Nearly all DNA polymerase add to the free 3'-OH group.

Anmerkungen:

• It has 2 replication forks with one origin of replication. Replication is semi discontinuous due to the one leading and lagging strand. This is lagging strand is called Okazaki's model of semi-discontinuous replication. Fragments are then joined by DNA ligase

1. Eurkayotic DNA

   Anmerkungen:

   • Difference is that they uses more DNA polymerases and have multiple sites of origins of replication

Anmerkungen:

• The semi-conservative model is the most accurate. I.e one side of a strand is the old one while the other side is the new one.

Anmerkungen:

• Eventually, there are some coding sites to terminate, it requires binding of TUS (Termination utilization substances)  to arrest the binding.

Anmerkungen:

• Telomerase extends chromosome ends because in the laggin strand, the DNA can't resynthesize and bind a nucleotide there. Hence, the telomerase adds a few when it was still able to do so, telomerase isn't present in adults in the same quantities as when it is a adolescent.

Anmerkungen:

• Damage is caused due to replication errors or external agents. It could lead to cancers, developmental abnormalities, mental retardation or ageing. Repair can be done by: Enzymes (base-pair errors) Excision (Large lumps) Or Recombination (join double stranded breaks)