Bacterial DNA repair

SCIENTIFIC AREA
Genome Dynamics and Function
CENTER
Centro de Biología Molecular “Severo Ochoa” CSIC-UAM (CBMSO)
VACANCIES
1
CONTACT E-MAIL
mdevega@cbm.csic.es
DESCRIPTION OF THE OFFER

The presence in PolXBs of AP endonuclease, 3´-5´ exonuclease, and DNA polymerase, three activities usually associated with different proteins that participate in Base excision repair, led to propose that bacterial PolXs could act as a backup mechanism for this repair pathway (Baños et al., 2008a, b; Baños et al., 2010). Interestingly, it has been recently reported that a B. subtilis strain lacking PolXBs was more sensitive to hydrogen peroxide than the parental isogenic strain; disruption of this gene significantly increased the susceptibility of an AP-endonuclease deficient strain to this oxidizing agent (Barajas-Ornelas et al., 2014). Importantly, during growth, the absence of PolXBs induced a significant increase in the level of mutant reversion (Barajas-Ornelas et al., 2014), in good agreement with the high nucleotide insertion fidelity exhibited by PolXBs (Baños et al., 2008a). However, the results also revealed that the genetic inactivation of PolXBs in the AP-endonuclease deficient genetic background significantly reduced the production of revertants in the stationary phase (Barajas-Ornelas et al., 2014). Authors speculated that the accumulation of AP sites in non-growing cells that overwhelms the capacity of the AP-endonucleases Nfo, ExoA and/or Nth promotes adaptive mutations following a mechanism that involves PolXBs processing of AP sites in an error-prone manner (Barajas-Ornelas et al., 2014). The question that arises then is how the same DNA polymerase performs faithful DNA polymerization during the exponential growth, and error-prone nucleotide insertion in the stationary phase.

It has been described that firmicutes, a bacterial phylum to which B. subtilis belongs, uptake Mn2+ ions late in the stationary phase and during the sporulation, the final concentration in the spore being much higher than that required during the exponential growth (Granger et al., 2011; Inaoka et al., 1999). It has been speculated that, in this case, the Mn2+ could be playing a protective role against oxidative damage most probably by binding and protecting the catalytic active sites of enzymes (Granger et al., 2011). We have previously shown that Mn2+ can be used as metal activator of the polymerization and AP-endonuclease activities of PolXBs (Baños et al., 2008a, b; Baños et al., 2010). Additionally, we have recently reported that PolXBs, unlike most DNA polymerases, exhibits a preferential incorporation of 8oxodGMP (the major dNTP lesion resulting from oxidative stress) opposite dC in 1nt-gapped molecules in the presence of Mg2+ (Zafra et al., 2017). Based on those precedents, it is tempting to hypothesize that the nucleotide insertion fidelity of PolXBs during the gap-filling step of BER, as well as its ability to insert the promutagenic 8oxodGMP syn conformation opposite dA could be influenced by the metal ion that is present late in stationary phase. To test this hypothesis, the analysis of both, nucleotide insertion fidelity and 8oxodGMP incorporation using Mn2+ as metal activator will be evaluated.

The ability of PolXBs to insert the 8oxodGMP in its (non)mutagenic conformation opposite dC and dA, respectively, will be analyzed by performing steady-state kinetic analyses incubating an overexcess of a 5´-Cy5 labeled 1 nt gapped DNA with the enzyme and increasing concentrations of the nucleotide in the presence of Mn2+ as metal activator.

Similarly, nucleotide insertion fidelity assays will be performed to infer the catalytic constants for the incorporation of the correct and wrong nucleotide. To avoid any interference of the 3´-5´ exonuclease activity, the above-mentioned studies will be carried out using the exonuclease deficient double mutant H339A/H341A (Baños et al, 2008b)

References

Baños, B., Lázaro, J.M., Villar, L., Salas, M., and de Vega, M. (2008a). J. Mol. Biol. 384, 1019-1028.

Baños, B., Lázaro, J.M., Villar, L., Salas, M., and de Vega, M. (2008b). Nucleic Acids Res. 36, 5736-5749.

Baños, B., Villar, L., Salas, M., and de Vega, M. (2010). Proc. Natl. Acad. Sci. U. S. A. 107, 19219-19224.

Barajas-Ornelas, R.C., Ramirez-Guadiana, F.H., Juarez-Godinez, R., Ayala-Garcia, V.M., Robleto, E.A., Yasbin, R.E., and Pedraza-Reyes, M. (2014). J. Bacteriol. 196, 3012-3022.

Granger, A.C., Gaidamakova, E.K., Matrosova, V.Y., Daly, M.J., and Setlow, P. (2011). Appl. Environ. Microbiol. 77, 32-40.

Inaoka, T., Matsumura, Y., and Tsuchido, T. (1999). J. Bacteriol. 181, 1939-1943.

Zafra, O., Pérez de Ayala, L., and de Vega, M. (2017). DNA Repair (Amst) 52, 59-69.

MASTER
Biomolecules & Cell D.
Molecular Biomedicine
Biotechnology