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Intrinsic properties of the two replicative DNA polymerases of Pyrococcus abyssi in replicating abasic sites: possible role in DNA damage tolerance?
Author(s) -
Palud Adeline,
Villani Giuseppe,
L'Haridon Stéphane,
Querellou Joël,
Raffin JeanPaul,
Henneke Ghislaine
Publication year - 2008
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2008.06446.x
Subject(s) - biology , dna polymerase , ap site , exonuclease , dna damage , dna , microbiology and biotechnology , dna clamp , dna repair , oligonucleotide , dna polymerase i , dna replication , sos response , polymerase , biochemistry , gene , polymerase chain reaction , reverse transcriptase
Summary 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, Pab polB and Pab polD, were characterized in presence of DNA containing abasic sites. Both Pab pols had abortive DNA synthesis upon encountering AP sites. Under running start conditions, Pab polB 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 Pab polD 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 Pab pols are high‐fidelity DNA polymerases onto undamaged DNA. Moreover, Pab pols 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 Pab pols. Our results are interpreted in terms of DNA damage tolerance.