enzyme is used to ligate the insert DNA into plasmid vector (An et al., 2010).
This job of sealing nicks (specifically, nicks that occur between a sugar 3' -OH group and the adjacent phosphate attached to the preceding sugar's 5' -OH group) is the function of DNA ligase
. Using a molecular energy source (which differs depending on the enzyme source organism), DNA ligase
reforms the missing covalent bond and the strand is whole again.
Mutants of the critical end-joining proteins XRCC4 and DNA ligase
IV have the most severe phenotype, both displaying V(D)J recombination defects, impaired lymphocyte development, p53-dependent neuronal apoptosis, and embryonic lethality [77-79].
BER has 4 main steps (base removal, apurinic/apyrimidinic (AP) site incision, synthesis, and ligation) and involves 4 major classes of DNA repair enzymes: DNA glycosylases, apurinic/apyrimidinic endonucleases (APEs), DNA polymerases (DNA pols), and DNA ligases
. Mammalian PARP-1 is a nuclear chromatin-associated multifunctional enzyme .
Finally, DNA ligase
catalyses the formation of two new phosphodiester bonds and thus seals the sugar-phosphate backbone."
III promotes alternative nonhomologous end-joining during chromosomal translocation formation.
(1) In NHEJ in vertebrate cells, seven molecules playing pivotal role have been identified: Ku70, Ku86 (also known as Ku80), DNA-PKcs (DNAdependent protein kinase catalytic subunit), Artemis, XRCC4, DNA ligase
IV (Lig4) and XRCC4-like factor (XLF, also known as Cernunnos).
This is analogous to that of PCNA, FEN-1, Pol[delta], and DNA ligase
1 being the main players for Okazaki fragment processing in eukaryotes [53, 62].
The digested DNA was ligated with T4 DNA ligase
to a final volume of 7.25[micro]L.
Next, 10[micro]L of a solution containing 1[micro]g of EcoRI and Msel adapters, 1X T4 DNA ligase
buffer and 1U of T4 DNA ligase
The main reagents used in the present study were AxyPrep-96 whole blood genomic DNA (AXYGEN, USA), Taq DNA polymerase (Qiagen Hotstar, Germany), Taq DNA ligase
enzyme (NEB, UK), and dNTP (Promega, USA).
Ligation was performed by the "ready to go" T4 DNA ligase
(Amersham Pharmacia Biotech).