Ligation Flashcards
What are DNA ligases?
DNA ligases represent a fundamental class of enzymes required by all organisms to maintain the structural integrity of the genome.
What cellular processes lead to breaks in the phosphate backbone structure of the DNA?
DNA replication, recombination and repair.
What do breaks in the phosphate backbone structure of the DNA lead to?
They can compromise the stability of the genome and threaten the loss of genetic information and introduction of deleterious chromosomal mutations.
What does DNA ligase do in replication process?
DNA replication requires the joining of Okazaki fragments (the small, nascent ssDNA fragments generated from the copying of the minus strand) by DNA ligase.
How are DNA breaks ultimately repaired through the catalytic activity of DNA ligases?
DNA ligases form a phosphodiester bond between adjacent nucleotides in duplex DNA.
What is the function of DNA ligases?
DNA ligases catalyze the formation of a phosphodiester bond between DNA single strands in the duplex form.
What type of bond does DNA ligase catalyze the formation of and between which two molecules?
DNA ligase catalyzes the formation of an internucleotide ester bond between phosphate and deoxyribose.
What 2 process take place when DNA ligase performs its function?
1) Covalent linkage between 5’ - P group of one chain with the adjacent 3’ - OH group of another.
2) Phosphate hydrolyses of the cofactor NAD or ATP.
What is needed for the ligase reaction to take place?
Energy is needed for a ligase reaction by the hydrolysis of ATP or NAD.
Which is more efficient the linkage of two DNA molecules with complementary sticky ends or blunt-end ligation?
The linkage of two DNA molecules with complementary sticky ends is more efficient.
What problem arises with blunt-end ligation?
Ligation reaction of a blunt-end DNA fragment into a plasmid also with blunt ends will result primarily in empty plasmids due to self ligation.
How do we solve the problem of self ligation?
By treating the plasmid with blunt ends with alkaline phosphatase enzyme which prevents self ligation.
What are the different types of DNA ligases?
1) Bacterial DNA ligases.
2) Bacteriophage DNA ligases.
3) Mammalian or eukaryotic DNA ligases.
4) Thermostable DNA ligases.
Mention ligases which use the hydrolysis of NAD as their energy source.
Bacterial DNA ligases, e.g., from E. coli, B. subtilis, and S. typhimurium.
Mention ligases which use the hydrolysis of ATP as their energy source.
Bacteriophages (e.g., T4 and T7) and eukaryotic cells.
