Structural evidence for an in trans base selection mechanism involving Loop1 in polymerase µ at an NHEJ double-strand break junction.

Eukaryotic DNA polymerase (Pol) X family members such as Pol µ and terminal deoxynucleotidyl transferase (TdT) are important components for the nonhomologous DNA end-joining (NHEJ) pathway. TdT participates in a specialized version of NHEJ, V(D)J recombination. It has primarily nontemplated polymerase activity but can take instructions across strands from the downstream dsDNA, and both activities are highly dependent on a structural element called Loop1. However, it is unclear whether Pol µ follows the same mechanism, because the structure of its Loop1 is disordered in available structures. Here, we used a chimeric TdT harboring Loop1 of Pol µ that recapitulated the functional properties of Pol µ in ligation experiments. We solved three crystal structures of this TdT chimera bound to several DNA substrates at 1.96-2.55 Å resolutions, including a full DNA double-strand break (DSB) synapsis. We then modeled the full Pol µ sequence in the context of one these complexes. The atomic structure of an NHEJ junction with a Pol X construct that mimics Pol µ in a reconstituted system explained the distinctive properties of Pol µ compared with TdT. The structure suggested a mechanism of base selection relying on Loop1 and taking instructions via the in trans templating base independently of the primer strand. We conclude that our atomic-level structural observations represent a paradigm shift for the mechanism of base selection in the Pol X family of DNA polymerases.

RNA mimicry by the fap7 adenylate kinase in ribosome biogenesis.

During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53-MDM2 pathway. This work presents the functional and structural characterization of the Fap7-Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.

A third purine biosynthetic pathway encoded by aminoadenine-based viral DNA genomes.

Cells have two purine pathways that synthesize adenine and guanine ribonucleotides from phosphoribose via inosylate. A chemical hybrid between adenine and guanine, 2-aminoadenine (Z), replaces adenine in the DNA of the cyanobacterial virus S-2L. We show that S-2L and Vibrio phage PhiVC8 encode a third purine pathway catalyzed by PurZ, a distant paralog of succinoadenylate synthase (PurA), the enzyme condensing aspartate and inosylate in the adenine pathway. PurZ condenses aspartate with deoxyguanylate into dSMP (N6-succino-2-amino-2'-deoxyadenylate), which undergoes defumarylation and phosphorylation to give dZTP (2-amino-2'-deoxyadenosine-5'-triphosphate), a substrate for the phage DNA polymerase. Crystallography and phylogenetics analyses indicate a close relationship between phage PurZ and archaeal PurA enzymes. Our work elucidates the biocatalytic innovation that remodeled a DNA building block beyond canonical molecular biology.

Terminal deoxynucleotidyltransferase: the story of an untemplated DNA polymerase capable of DNA bridging and templated synthesis across strands.

Terminal deoxynucleotidyltransferase (TdT) is a member of the polX family which is involved in DNA repair. It has been known for years as an untemplated DNA polymerase used during V(D)J recombination to generate diversity at the CDR3 region of immunoglobulins and T-cell receptors. Recently, however, TdT was crystallized in the presence of a complete DNA synapsis made of two double-stranded DNA (dsDNA), each with a 3' protruding end, and overlapping with only one micro-homology base-pair, thus giving structural insight for the first time into DNA synthesis across strands. It was subsequently shown that TdT indeed has an in trans template-dependent activity in the presence of an excess of the downstream DNA duplex. A possible biological role of this dual activity is discussed.

Structural Basis for a New Templated Activity by Terminal Deoxynucleotidyl Transferase: Implications for V(D)J Recombination.

Eukaryotic DNA polymerase of the polX family, such as pol µ and terminal deoxynucleotidyl transferase (TdT), are key components of the non-homologous end-joining or V(D)J recombination machinery, respectively. The established role of TdT is to add random nucleotides during V(D)J recombination. Here we show that TdT also has a templated-polymerase activity, similar to pol µ, in the presence of higher concentrations of a downstream DNA duplex, and performs a micro-homology single base-pair search to align the DNA synapsis. To understand the molecular basis of this alignment, we solve crystal structures of TdT with four DNA strands and study the influence of the 3' protruding end. Two mutations in TdT inspired by sequence alignments with pol µ further improve the templated activity. We propose that both templated and untemplated activities of TdT are needed to explain the distributions of lengths of N regions observed experimentally in T cell receptors and antibodies.