Lecture 16 Flashcards
(27 cards)
Copying DNA
- Separating DNA strands.
- Primers bind to complementary sequence. The 3’ OH is needed for the addition of nucleotides
- Nucleotide added to strand via DNA Polymerase. H bond formed between complementary bases. Polymerase forms phosphodiester bond between backbone.
- Depending on DNA polymerase used, sequence may be proofread.
DNA read 3’-5’ continues until end of strand reached.
E.Coli
Grows quickly. Genome needs to be copied everytime it replicates. DNA is circular.
DnaA
Recognises origin site on E.Coli. Separates DNA
DnaB (helicase)
Loaded to each strand. Moves 5’-3’ to unwind DNA. Moves in anti-parallel, they move opposite to each other.
Single-stranded-DNA-binding-protein (ssB)
Keeps DNA separated.
E.Coli Primase
Bind to (or rather, synthesised on) complementary sequence. All DNA polymerases need a primer to add nucleotides to. Provides the 3’ OH the nucleotide is added to.
Primase (DnaG)
RNA polymerase does not need primers for synthesis. DNA polymerase uses this RNA primer for DNA synthesis.
Nucleoside
Base and sugar
Check 1
Before adding the nucleotide, is the base pairing correct? If incorrect, visually it fits poorly in the active site and hopefully the nucleotide dissociates away from strand.
nucleotide
Nucleoside and phosphate. Substrate for nucleic acid synthesis. Generalised to NTPs or dNTPs (RNA or DNA dependent)
Active site of DNA polymerase
1: Base pairing with template
2: DNA polymerase catalyses formation of phosphodiester bond between terminal phosphate and O.
3: Pyrophosphate released and breaks down to 2P.
Thermostability of DNA/RNA synthesis
DNA/RNA synthesis is thermodynamically unfavourable. Free energy released fro, phosphates in DNA synthesis.
Check 2
Double check added nucleotide. If not, cut off by 3’ to 5’ exonuclease.
Nucleases
3’-5’ Exonuclease cut from 3’ end
5’-3’ Exonuclease cut from 5’ end
Endonuclease cut in between
Replication fork
Replication bubble grows as helicase moves along each strand at each replication fork as it opens.
Lagging strand
Synthesised at Okazaki fragments.
DNA Polymerase III
9 subunits. Pol. III core adds nucleotides and proofreads. B sliding clamp holds tightly onto DNA and synthesis stops when it disassociates (fall off). Clamp loader grabs the lagging strand and moves it to the polymerase.
Supercoiling
As DNA is separated by helicase, the part infront of it (unwound part) starts to supercoil.
Removing Primers
DNA Polymerase I removes RNA primers due to its 5’→3’ exonuclease activity. At the same time, its polymerase activity adds DNA nucleotides to fill in the gap. However, DNA Pol I cannot form the final phosphodiester bond between the new DNA and the adjacent DNA fragment.Done by DNA ligase which forms the final phosphodiester bond
Positive supercoiling
Twisting in same direction as helix (tighter/harder to separate)
Negative supercoiling
Twist in opposite direction to helix (easier to separate)
Reaching the end
Important that genome is copied once. There are ‘traps’ for the fork if it approaches from other direction. Another topoisomerase separates chromosomes
DNA topoisomerase II
Introduces negative supercoils as helicase opens DNA. Needs ATP.
Acyclovir (antiviral drug)
Treats herpes simplex virus. Nucleoside analogue (modified nucleoside). No sugar. Viral thymidine kinase phosphorylates drug form while host does not. It is incorporated into infected cell DNA, preventing further synthesis as there’s no 3’ OH to add nucleotides.
