DNA Metabolism Enzymes Flashcards
Proteins generally recognise sequence dependent variations in DNA structure. During the past 15 years high resolution structures of many protein DNA complexes have been solved, what similar structural domains are present in these proteins?
Zinc fingers, Zinc binding domains, Helix-turn-helix domains, Basic region Leucine zipper, Beta sheet recognition domain.
The DNA helix can take three different forms, what are these forms called and what orientation are they?
A-helices are right handed,
B-helices are right handed,
Z-helices are left handed.
What unusual DNA structures are there?
Hairpins and cruciforms- inverted repeat sequences can form cruciforms.
Tetraplexes- these are 4-stranded DNA structures.
What are the three stages of DNA replication?
What is the main enzyme responsible for this process?
What type if conservation is DNA replication?
What is the direction of DNA replication?
Initiation, elongation and termination.
DNA polymerase is the main enzyme responsible.
DNA replication is semi conservative.
Direction is 5’ to 3’
Describe DNA ligases.
Hesse enzymes are critical for the final step of DNA synthesis. Eukaryotic ligases use ATP as a cofactor, bacterial ligases use NAD^+.
All bacteria contain genes for NAD^+ dependent ligases.
The enzyme works by locating the DNA break, opening, closing around the DNA, lighting the break and the opening to release the DNA.
What are RNA ligases?
These have been identified as a separate class of enzymes that can join together some nucleic acid molecules. They appear to be more involved with the repair, splicing and editing pathways of RNA. The best characterised RNA ligases are from the T4 bacteriophage.
How can DNA ligases be used as therapeutic targets?
Defects in specific DNA ligases have been identified in several human diseases, including cancer and neurodegradation.
Enzymes that are essential in bacteria and which function differently could be targeted by antibiotics.
Give two examples of bacterial DNA ligase inhibitors and one example of a human DNA ligase inhibitor.
Bacterial ligase inhibitors- glycosyl amines,
Glycosyl ureides
Human ligase inhibitors- anthracycle analogues inhibit human ligases.
What are the limitations of electrophoretic assays?
There is not a high throughput which is necessary if DNA ligases are to be studied as targets for potential therapies.
There are also mechanistic limitations which limit analysis of interactions between various compounds of a reaction.
What are immobilised DNA hairpin assays?
These use DNA hairpins with nicks that are immobilised on microtitre plates though a biotin-streptavidin linkage and are also covalently linked to a fluorophore at the 5’ end. When in an assay of ligase if the ligase isn’t functional of the DNA hairpin, the nicked DNA will be washed off and there will be no fluorescence. If the ligase is functional then it will not be washed off and thus there will be fluorescence.
What processes are involved in DNA metabolism?
DNA recombination,
DNA repair,
DNA replication,
DNA transcription.
