Question VI. In 1976, Hozumi and Tonegawa published an elegant paper (Proc. Natl. Acad. Sci. USA 73, 3628-3632, 1976) showing that a V gene and a C gene are miles apart in germline DNA (from mouse embryos), but next to each other in antibody-coding DNA (from a myeloma tumor). Subsequent experiments have demonstrated unequivocally that the immune system generates the antibody diversity by shuffling a few hundred genes to create countless permutations, in a process called V(D)J recombination. In this process, RAG enzymes work as a multi-subunit complex to induce cleavage of a double-stranded DNA molecule between the antigen receptor coding segment and a flanking recombination signal sequence (RSS). They do this in two steps. They initially introduce a ‘nick’ in the 5\' (upstream) end of the RSS heptamer (a conserved region of 7 nucleotides) that is adjacent to the coding sequence, leaving behind a 3\'-hydroxyl group at the coding end and a 5\'-phosphate group at the RSS end. The next step couples these chemical groups, binding the OH-group (on the coding end) to the PO4group (that is sitting between the RSS and the gene segment on the opposite strand). [Assess each subsequent statement (64-71) as either factually correct (= “true”) or incorrect (= “false”)]: 64. This is an important enzymatic step in a process known as somatic recombination, which is limited to macrophages. 65. This is an event of the mechanism of genetic recombination in the early stages of Ig and TCRs production. 66. This produces a 3\'-phosphorylated double-stranded break at the RSS and a covalently closed hairpin at the coding end. 67. This produces a 3\'-phosphorylated double-stranded break at the RSS and a non-covalently closed hairpin at the coding end. 68. This produces a 5\'-phosphorylated double-stranded break at the RSS and a covalently-closed hairpin at the coding end. 69. This produces a 5\'-phosphorylated double-stranded break at the RSS and a non-covalently closed hairpin at the coding end. 70. This is an event of the mechanism of genetic recombination in the late stages of immunoglobulin (Ig) and T-cell receptors (TCRs) production. 71. The V(D)J recombination events are involved in somatic hypermutations of the TCR and the binding of the OH-group (on the coding DNA end) to the PO4-group (that is sitting between the RSS and the gene segment on the opposite DNA strand) plays an important role in these hypermutations, which contribute to generating TCR diversity. Solution 64. False This process is not limited to macrophages and occurs in the B cells and T cells. 65. True This occurs in early stages of production of T cell receptors as well as immunoglobulins. 66. False No 3\'-phosphorylated double-stranded break is produced 67. False No 3\'-phosphorylated double-stranded break is produced 68. True Yes, 5\'-phosphorylated break is produced at the RSS as well as on the coding end a covalently closed- hairpin is formed. 69. False Non- covalently closed hairpin is not .

1. Question VI. In 1976, Hozumi and Tonegawa published an elegant paper (Proc. Natl. Acad. Sci.
USA 73, 3628-3632, 1976) showing that a V gene and a C gene are miles apart in germline DNA
(from mouse embryos), but next to each other in antibody-coding DNA (from a myeloma tumor).
Subsequent experiments have demonstrated unequivocally that the immune system generates the
antibody diversity by shuffling a few hundred genes to create countless permutations, in a
process called V(D)J recombination. In this process, RAG enzymes work as a multi-subunit
complex to induce cleavage of a double-stranded DNA molecule between the antigen receptor
coding segment and a flanking recombination signal sequence (RSS). They do this in two steps.
They initially introduce a ‘nick’ in the 5' (upstream) end of the RSS heptamer (a conserved
region of 7 nucleotides) that is adjacent to the coding sequence, leaving behind a 3'-hydroxyl
group at the coding end and a 5'-phosphate group at the RSS end. The next step couples these
chemical groups, binding the OH-group (on the coding end) to the PO4group (that is sitting
between the RSS and the gene segment on the opposite strand). [Assess each subsequent
statement (64-71) as either factually correct (= “true”) or incorrect (= “false”)]:
64. This is an important enzymatic step in a process known as somatic recombination, which is
limited to macrophages.
65. This is an event of the mechanism of genetic recombination in the early stages of Ig and
TCRs production.
66. This produces a 3'-phosphorylated double-stranded break at the RSS and a covalently closed
hairpin at the coding end.
67. This produces a 3'-phosphorylated double-stranded break at the RSS and a non-covalently
closed hairpin at the coding end.
68. This produces a 5'-phosphorylated double-stranded break at the RSS and a covalently-closed
hairpin at the coding end.
69. This produces a 5'-phosphorylated double-stranded break at the RSS and a non-covalently
closed hairpin at the coding end.
70. This is an event of the mechanism of genetic recombination in the late stages of
immunoglobulin (Ig) and T-cell receptors (TCRs) production.
71. The V(D)J recombination events are involved in somatic hypermutations of the TCR and the
binding of the OH-group (on the coding DNA end) to the PO4-group (that is sitting between the
RSS and the gene segment on the opposite DNA strand) plays an important role in these
hypermutations, which contribute to generating TCR diversity.
Solution

2. 64. False
This process is not limited to macrophages and occurs in the B cells and T cells.
65. True
This occurs in early stages of production of T cell receptors as well as immunoglobulins.
66. False
No 3'-phosphorylated double-stranded break is produced
67. False
No 3'-phosphorylated double-stranded break is produced
68. True
Yes, 5'-phosphorylated break is produced at the RSS as well as on the coding end a covalently
closed- hairpin is formed.
69. False
Non- covalently closed hairpin is not formed. Instead a covalently closed hairpin is formed.
70. False
This occurs in early stages and not in late stages.