Intrinsic properties of the two replicative DNA polymerases of Pyrococcus abyssi in replicating abasic sites: possible role in DNA damage tolerance?
|Author(s)||Palud Adeline1, 2, Villani Giuseppe3, L'Haridon Stephane2, Querellou Joel1, 2, Raffin Jean-Paul1, 2, 4, Henneke Ghislaine1, 2|
|Affiliation(s)||1 : Inst Francais Rech Exploitat Mer, UMR 6197, Lab Microbiol Environm Extremes, F-29280 Plouzane, France.
2 : Univ Bretagne Occidentale, UMR 6197, Lab Microbiol Environm Extremes, F-29280 Plouzane, France.
3 : Univ Toulouse 3, CNRS, Inst Pharmacol & Biol Struct, UMR 5089, F-31077 Toulouse, France.
|Source||Molecular Microbiology (0950-382X) (Wiley / Blackwell), 2008-11 , Vol. 70 , N. 3 , P. 746-761|
|WOS© Times Cited||23|
|Abstract||Spontaneous and induced abasic sites in hyperthermophiles DNA have long been suspected to occur at high frequency. Here, Pyrococcus abyssi was used as an attractive model to analyse the impact of such lesions onto the maintenance of genome integrity. We demonstrated that endogenous AP sites persist at a slightly higher level in P. abyssi genome compared with Escherichia coli. Then, the two replicative DNA polymerases, PabpolB and PabpolD, were characterized in presence of DNA containing abasic sites. Both Pabpols had abortive DNA synthesis upon encountering AP sites. Under running start conditions, PabpolB could incorporate in front of the damage and even replicate to the full-length oligonucleotides containing a specific AP site, but only when present at a molar excess. Conversely, bypassing activity of PabpolD was strictly inhibited. The tight regulation of nucleotide incorporation opposite the AP site was assigned to the efficiency of the proof-reading function, because exonuclease-deficient enzymes exhibited effective TLS. Steady-state kinetics reinforced that Pabpols are high-fidelity DNA polymerases onto undamaged DNA. Moreover, Pabpols preferentially inserted dAMP opposite an AP site, albeit inefficiently. While the template sequence of the oligonucleotides did not influence the nucleotide insertion, the DNA topology could impact on the progression of Pabpols. Our results are interpreted in terms of DNA damage tolerance.|