26 | Chapter 2 More detailed studies of the initiation of V(D)J recombination revealed that DNA melting of the conserved RSS heptamer proceeds the nicking and suggested that the conservation of the heptamer is determined by its ability to unwind at CAC/GTG sequences73. Subsequently, trans-esterification takes place during which the nicked 3’OH of the coding strand invades the opposite DNA strand, creating a DSB, which contains a closed hairpin at the coding end and a blunt 5’RSS end (signal end). RAG1 and RAG2 are supported by high-mobility group proteins belonging to the HMG-box family (HMGB1 and HMGB2), which facilitate the association of two signal ends. The HMG proteins interact with the nonamer-binding domain of RAG1 even in the absence of DNA, amplifying its natural DNA-binding activity74. Following the DNA cutting, Ku70 (also known as X-ray repair cross-complementing group 5 or XRCC5) and Ku80 (also known as X-ray repair cross-complementing group 6 or XRCC6) heterodimerize and bind the broken DNA ends and attract DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which controls the interaction of the broken DNA ends. The Ku70/Ku80 heterodimer also attracts other factors containing Ku-binding motifs75,76. Even though they form a heterodimer, striking differences in their mutant phenotypes have been observed, while the ku80-/- mice were reported to show early aging signs without the increased incidence of cancer, the ku70-/- mice exhibited defective B-cell maturation and high incidence of thymic lymphomas77. Silencing of Ku70 in the Jurkat T-cell leukemia cell line resulted in accumulation of DSBs, cell cycle arrest, and increased apoptosis as a consequence of the impairment of the DNA repair pathways by the Ku70 deficiency78. Next, from the four broken DNA ends two are covalently sealed forming a hairpin (termed “coding end”), and the other two are blunt DNA ends (termed “signal ends”). For this reaction no external energy is required, the necessary energy is derived from the DNA breakage. The binding of RAG1/2 complex to the DNA does not form a covalent complex and while the nicking occurs within minutes, the hairpinning might take several hours79. Subsequently, the hairpins must first be opened in order to proceed with the joining and repair of the broken DNA. A characteristic feature of V(D)J recombination is the asymmetric processing of the signal and coding end. The blunt-ended signal ends can directly be ligated with almost no processing, while the coding ends must first undergo further processing, such as small deletions or small insertions80,81. In the process of V(D)J recombination, Artemis is a nuclease with an indispensable role in resolving and repairing the DSBs. Artemis exhibits an inherent 5’-3’exonuclease activity while upon an association with DNA-PKcs, Artemis acts as a 5’-3’endonuclease. The hairpin is opened asymmetrically on the coding end, creating a shorter and a longer strand. The shorter strand is extended by the addition of nucleotides complementary to the longer strand, giving rise to the insertion of palindromic nucleotides (P-nucleotides) at the coding end. These are never observed at the signal end. In addition, the single-strand extensions increase the chances of loss of nucleotides from the coding end, thereby further increasing the diversity of the
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