1. Structural Basis for a New Templated Activity by Terminal Deoxynucleotidyl Transferase: Implications for V(D)J Recombination 
Jérôme Loc'h, Sandrine Rosario, Marc Delarue 
Structure 
Volume 24, Issue 9, Pages (September 2016)
DOI: /j.str
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2. Structure 2016 24, 1452-1463DOI: (10.1016/j.str.2016.06.014)
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3. Figure 1 Functional Tests of Mouse TdT with a Downstream DNA Duplex
To mimic a DSB-DNA substrate, a mixture of a radiolabeled (upstream) primer strand is mixed with a downstream DNA duplex whose 3′-protruding end can make one micro-homology base pair (MH-bp) (in red) with the last 3′ base of the primer strand. The templating base X is in blue. The (-TdT) lane is the control lane without added TdT and the four other lanes correspond to each specific dNTP added (A, G, C, T). The samples were analyzed by gel electrophoresis on a 15% acrylamide gel with 8 M urea.
(A) TdT (10 nM) activity with various amounts of a downstream duplex substrate (2–200 nM) that can make a Watson-Crick MH-bp (in red).
(B) TdT (10 nM) activity in the presence of various downstream duplexes (200 nM) with four different templating bases and the same MH-bp.
(C) Pol μ (200 nM) and downstream duplexes (200 nM) with four different templating bases and the same MH-bp (in red).
(D) TdT (10 nM) and downstream duplexes (200 nM) with three different non-Watson-Crick base pairs at the MH position (in red).
Structure  , DOI: ( /j.str )
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4. Figure 2
Structures of Four-Stranded DSB-DNA Substrate Bound to Mouse TdT
(A) DNA substrates: the upstream primer strand is colored in red, its based-paired template strand is in yellow, and the downstream DNA duplex is in blue and cyan. Loop1 is depicted in green.
(B) The complete DSB-TdT structure in two situations: (left) with a T5GC primer (one MH-bp and one Watson-Crick nascent base pair) and (right) a T5GGC primer: the extra base causes one of the bases to loop out (bulge) of the DNA mini-helix path so as to keep the MH-bp.
(C) Stabilization of the MH mini-helix region in the T5GC and T5GGC complexes. L398 and F405 side chains are shown in ball-and-stick representation. D396 side chain undergoes a major conformational change in the absence/presence of the 5′ extra base of the upstream template strand (yellow), while the R393 side chain becomes ordered. Loop1 is interrupted in the electron density and a tentative pathway that links the broken ends is indicated as a green dashed line to help read the main-chain path. Black dashed lines represent hydrogen bonds.
(D) Details of the R393 side-chain stacking interactions with the extra base of the downstream template strand in the T5GGC complex.
(E) Recognition of the G unpaired base of the primer strand by an aspartate side chain D396 in the T5GGC complex. The standard C-G base pair in Watson-Crick geometry is shown in transparency. The red sphere represents the water molecule closest to the guanosine.
Structure  , DOI: ( /j.str )
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5. Figure 3
Protein-DNA Interactions between Mouse TdT and the Synaptic DNA-DSB Substrate: DNA Quadruplex
(A) Schematic representation of the DNA-protein interactions. Two situations are represented: without (left) and with (right) a single-nucleotide bulge, with primer strands T5GC and T5GGC, respectively. The primer strand is in red and the upstream template strand is in yellow. The downstream DNA duplex is in cyan and blue.
(B) Interactions of the upstream template strand (yellow) with the protein in T5GC (left) and T5GGC (right) complexes. Loop1 is in green.
(C) Same view in the gap-filling complex of pol μ (PDB: 4YD1). Most of Loop1 is disordered.
Structure  , DOI: ( /j.str )
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6. Figure 4
Loop1 Recognition of Four Different 3′ Protruding Ends in the DNA-DSB Site: DNA Synapsis
Only the MH mini-helix DNA region is represented. In all cases, the L398 and F405 side chains are ordered and induce a wedge in the path of the phosphate backbone of the primer strand. Loop1 is in green and the important side chains of the protein are represented in ball-and-stick and numbered. The primer strand is in red.
(A) Characteristics of the four DNA substrates of TdT complexes solved in this study.
(B) T5GC primer strand, showing two base pairs: the MH-bp and the nascent base pair.
(C) T5GGC primer strand, also showing the same two base pairs but with an extra base bulging out of the in trans template strand.
(D) First complex obtained with the A5C primer strand, with only the nascent base pair visible in the electron density.
(E) Second complex obtained with the A5C primer strand, with no visible nascent base pair.
Structure  , DOI: ( /j.str )
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7. Figure 5
Functional Tests of Wild-Type and Mutants of Mouse TdT in the Presence of a Downstream DNA Duplex
The DNA substrate contains a radiolabeled (upstream) primer strand mixed with a downstream DNA duplex whose 3′ protruding end can make one MH-bp (in red) with the last 3′ base of the primer strand. The (in trans) templating base X is in blue. The (-TdT) lane is the control lane without added TdT and the four other lanes correspond to each specific dNTP added (A, G, C, T). The samples were analyzed by gel electrophoresis on a 15% acrylamide gel with 8 M urea.
(A) TdT (200 nM) activity in the presence of various downstream duplexes (200 nM) with four different templating bases and the same MH-bp (in red).
(B) S187R and D179A TdT mutants (200 nM) and downstream duplexes (200 nM) with four different templating bases and the same MH-bp.
(C) TdT Loop1-pol μ mutant (200 nM) and downstream duplexes (200 nM) with four different templating bases and the same MH-bp.
Structure  , DOI: ( /j.str )
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8. Figure 6
Extension to Other polX that Bind DNA DSB: The Case of a Downstream Template Strand Containing One Looped-Out Base
(A) Sequence multi-alignment of mouse TdT, an alternative splicing form of mouse TdT, mouse pol μ, yeast pol IV, human pol λ, and human pol β. Loop1, Loop2, and Loop3 are highlighted as well as SD1 and SD2 (purple and blue boxes, respectively). Catalytic aspartate residues are underlined by a red dot. Strictly conserved residues are highlighted in red.
(B) Structural alignment of template strands of mouse TdT (PDB: 5D49, in gray) in the DSB complex, mouse pol μ (PDB: 4YD1, in orange), and human pol λ (PDB: 2BCV, in green) in a gap-filling complex. The conformations of Loop1 of TdT and Loop3 of pol λ are represented in gray and green, respectively. SD1 and SD2 regions are represented in purple and blue, respectively.
Structure  , DOI: ( /j.str )
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9. Figure 7 Model of TdT Activity during V(D)J Recombination
(A) The V(D)J complex when Artemis (purple) generates a DSB DNA by opening the hairpins left over after the action of Rag1/Rag2. DNA-PKcs is sketched in pink.
(B) Growing phase for the N regions during untemplated TdT activity. Two 3′ protruding bases have been drawn for the illustration, instead of the four left by Artemis immediately after opening the hairpins, assuming a limited exonuclease activity of Artemis. TdT is in green, Ku 70/80 is schematized in light blue, and the incoming nucleotide is in red.
(C) After typically four nucleotide incorporations (in red), i.e., a length extension of the primer strand of about 15 Å, TdT binds the downstream duplex and its activity becomes (in trans) templated, and eventually comes to a halt for a lack of cognate MH-bp.
Structure  , DOI: ( /j.str )
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