Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom

Abstract

The catalytic active site of the Polymerization Domain (PolDom) of bacterial Ligase D is designed to promote realignments of the primer and template strands and extend mispaired 3′ ends. These features, together with the preferred use of ribonucleotides (NTPs) over deoxynucleotides (dNTPs), allow PolDom to perform efficient double strand break repair by nonhomologous end joining when only a copy of the chromosome is present and the intracellular pool of dNTPs is depleted. Here, we evaluate (i) the role of conserved histidine and serine/threonine residues in NTP insertion, and (ii) the importance in the polymerization reaction of a conserved lysine residue that interacts with the templating nucleotide. To that extent, we have analyzed the biochemical properties of variants at the corresponding His651, Ser768, and Lys606 of Pseudomonas aeruginosa PolDom (Pa-PolDom). The results show that preferential insertion of NMPs is principally due to the histidine that also contributes to the plasticity of the active site to misinsert nucleotides. Additionally, Pa-PolDom Lys606 stabilizes primer dislocations. Finally, we show that the active site of PolDom allows the efficient use of 7,8-dihydro-8-oxo-riboguanosine triphosphate (8oxoGTP) as substrate, a major nucleotide lesion that results from oxidative stress, inserting with the same efficiency both the anti and syn conformations of 8oxoGMP.

The enterohemorrhagicEscherichia coliinsertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity

“…As mentioned above, the catalytic active site of extensively characterized AEP-containing proteins such as PaeLigD, MtuLigD and PrimPolC, includes a histidine residue (PaeLigD H651, MtuLigD H111 and PrimPolC H122) that contacts the ribose 2’-OH group of the incoming ribonucleotide [(37–39), see also the sequence alignment in Supplementary Figure S1 ]. Site-directed mutagenesis at the corresponding PaeLigD H651 allowed the authors to conclude that this residue is the main structural element responsible for the discrimination against dNTP insertion exhibited by these enzymes ( 40 ). Other AEP members that proficiently discriminate against ribonucleotides, such as Pyrococcus furiosus p41 ( 50 ) and human PrimPol ( 51 ), have a tyrosine residue in its place (Y72 and Y100, respectively) ( 52 ).…”

LigD: A Structural Guide to the Multi-Tool of Bacterial Non-Homologous End Joining

“…The ability of the LigD POL domain to insert ribonucleotides preferentially is of interest ( Della et al, 2004 ; Pitcher et al, 2007a ; Zhu and Shuman, 2010 ). Biochemical studies in P. aeruginosa show that the His111 homolog, when mutated in P. aeruginosa LigD, has reduced preference for ribonucleotides compared to wild-type LigD ( Sánchez-Salvador and De Vega, 2020 ). In both tripartite complex structures from M. tuberculosis and P. aeruginosa , the conserved histidine hydrogen bonds to the 2′-hydroxyl of the ribose, dictating selectivity for ribonucleotides in the active site ( Sánchez-Salvador and De Vega, 2020 ).The POL domain structure also plays a role in maintaining fidelity.…”

“…Biochemical studies in P. aeruginosa show that the His111 homolog, when mutated in P. aeruginosa LigD, has reduced preference for ribonucleotides compared to wild-type LigD ( Sánchez-Salvador and De Vega, 2020 ). In both tripartite complex structures from M. tuberculosis and P. aeruginosa , the conserved histidine hydrogen bonds to the 2′-hydroxyl of the ribose, dictating selectivity for ribonucleotides in the active site ( Sánchez-Salvador and De Vega, 2020 ).The POL domain structure also plays a role in maintaining fidelity. P. aeruginosa LigD showed decreased polymerization efficiency and increased nucleotide misincorporation with a mutation of Lys606 to alanine ( Sánchez-Salvador and De Vega, 2020 ).…”

“…In both tripartite complex structures from M. tuberculosis and P. aeruginosa , the conserved histidine hydrogen bonds to the 2′-hydroxyl of the ribose, dictating selectivity for ribonucleotides in the active site ( Sánchez-Salvador and De Vega, 2020 ).The POL domain structure also plays a role in maintaining fidelity. P. aeruginosa LigD showed decreased polymerization efficiency and increased nucleotide misincorporation with a mutation of Lys606 to alanine ( Sánchez-Salvador and De Vega, 2020 ). This lysine is conserved and in M. tuberculosis , the homologous Lys66 sandwiches the base of either the incoming nucleotide or templating DNA strand with Phe64 ( Figure 6B ) ( Pitcher et al, 2007b ; Brissett et al, 2011 ).…”

“…This lysine is conserved and in M. tuberculosis , the homologous Lys66 sandwiches the base of either the incoming nucleotide or templating DNA strand with Phe64 ( Figure 6B ) ( Pitcher et al, 2007b ; Brissett et al, 2011 ). While the exact mechanism of fidelity is unclear, it is hypothesized that the lysine helps select the correct nucleotide for insertion, when a DNA template is present ( Sánchez-Salvador and De Vega, 2020 ).…”