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

Alejandro Sánchez-Salvador,Miguel De Vega

doi:10.3390/biom10020203

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.

Highlights

DNA double-strand breaks (DSBs) are the most hazardous DNA lesions that have to be repaired in a timely fashion to prevent genome instability [1]
We show that the active site of Polymerization Domain (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 crystallographic structures of the PolDom of P. aeruginosa LigD (Pa-PolDom) with a Mn2+-ATP bound at the active site [31], as well as of the ternary complex of Mt-PolDom with an incoming UTP forming a Watson–Crick base pair with a templating adenine base [23] showed that the ribose 2 -OH group of the nucleotide is hydrogen bonded to the Nδ moiety of a conserved histidine residue (Pa-LigD His651, Mt-LigD His111; see Figure 1)

Summary

Introduction

DNA double-strand breaks (DSBs) are the most hazardous DNA lesions that have to be repaired in a timely fashion to prevent genome instability [1]. The two major DNA repair pathways to mend DSBs are homologous recombination (HR) and nonhomologous end-joining (NHEJ) (reviewed in [2]). Whereas HR relies on the presence of an intact chromatid copy used as the template for DNA synthesis across the break, NHEJ performs the direct ligation of the breaks, without needing a template strand. In the latter case, as the termini to be joined are often either non-compatible or are damaged, they are processed before the final ligation step, giving rise to insertions, deletions, and base substitutions at the break site, NHEJ being considered an error-prone pathway [2,3]. Non-compatible termini, as well as those containing lesions, are remodeled by the action of nucleases (Artemis and APLF) and of the family X DNA polymerases Pol λ, Pol μ, and TdT [3]

Methods

Results

Conclusion