Role of translesion DNA polymerases in mutagenesis and DNA damage tolerance in Pseudomonads
Date
2018-07-09
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Abstract
Kahjustused DNA-s, mis tekivad kas rakkude normaalse elutegevuse käigus või erinevate keskonnategurite mõjul (näiteks UV-kiirgus, DNA-d kahjustavad kemikaalid), pärsivad genoomi replikatsiooni, takistades replikatiivse DNA polümeraasi edasiliikumist. Pikaajaline replikatsiooni seiskumine võib osutuda rakkudele letaalseks. Selleks, et DNA replikatsioon saaks jätkuda ka kahjustatud DNA-lt, on välja kujunenud DNA kahjustuste tolereerimise mehhanismid. Üheks neist on DNA kahjustustest ülesüntees (translesion DNA synthesis, TLS), mida viivad läbi spetsialiseeritud DNA polümeraasid. Need polümeraasid jätkavad DNA sünteesi kahjustatud nukleotiidi kohalt, tagades organismi ellujäämise DNA kahjustuste olemasolul. Samas võib vigaderohke süntees viia mutatsioonide tekkeni, mis on alusmaterjaliks evolutsioonile, kuid põhjustavad ka geneetilisi haigusi. Näiteks bakteritel on TLS polümeraaside toimel tekkinud geneetiline variantsus oluline antibiootikumide resistentsuse ja infektsioonivõime kujunemisel. TLS polümeraasid on potentsiaalseks märklauaks nii antibakteriaalses ravis kui ka vähiteraapias.
Minu doktoritöö eesmärgiks oli selgitada TLS polümeraaside funktsioone, eeskätt nende võimalikku rolli mutatsiooniprotsessides ning DNA kahjustuste tolereerimisel perekonda Pseudomonas kuuluval mullabakteril P. putida ja inimese oportunistlikul patogeenil P. aeruginosa. Pseudomonaadidel on kolm TLS polümeraasi: Pol II, Pol IV ja ImuABC. Uurimistöö tulemused viitavad sellele, et bakteris P. putida võivad TLS polümeraasid osaleda DNA sünteesil DNA polümeraasi Pol I puudumisel. Lisaks selgus, et Pol IV ja ImuABC on olulised DNA alküülkahjustuste talumisel. Kui ImuC viib läbi vigaderohket sünteesi, suurendades mutatsioonide arvu, siis Pol IV ületab DNA alküülkahjustusi väga täpselt. Üllatuslikult selgus, et bakterite inkubeerimise temperatuur mõjutab DNA alküülkahjustuste tolereerimist ja/või reparatsiooni efektiivsust.
The integrity of our hereditary material is constantly challenged by both endogenous agents formed during normal cellular metabolism and by various exogenous factors, like UV-light and chemicals found everywhere in the environment. Damage in DNA can block genome replication, which can lead to genomic instability and death of the cell. To overcome these blocks, organisms have evolved DNA damage tolerance mechanisms that allow completion of DNA replication in the presence of damage. One of them is translesion DNA synthesis (TLS), which is mediated by specialized TLS DNA polymerases that are able replicate over the replication-blocking DNA damage. Information encoded by a damaged nucleotide is usually inaccurate, therefore TLS is inherently error-prone process. As such, TLS polymerases not only protect cells against DNA damage, but also represent a potential source of mutations – a material for evolution, but also a cause of disease in humans. In bacteria, genetic diversity generated by TLS polymerases is critical for the acquisition of antibiotic resistance. Therefore, a lot of research is now undertaken to use TLS polymerases as a target for cancer or antimicrobial treatment. In the present work, I describe the role of TLS polymerases in two representatives of Pseudomonas, important human pathogen Pseudomonas aeruginosa and soil bacterium Pseudomonas putida. These organisms possess three TLS polymerases: Pol II, Pol IV and ImuABC. The results of our work revealed that in P. putida TLS polymerases might leave mutagenic fingerprints in the genome during normal growth of bacteria, suggesting their potential role in bypass past endogenously formed DNA damage. Moreover, Pol II and Pol IV might be involved in DNA replication in the absence of replicative DNA polymerase I. Both Pol IV and ImuABC in Pseudomonads are involved in DNA alkylation damage tolerance. Replication across alkylation damage by ImuABC is highly mutagenic, Pol IV, on the contrary, performs very accurate bypass. One of the important findings of my study was that simple switch in growth temperature of bacteria changed the cell’s strategy to deal with alkylation DNA damage and role of TLS in it.
The integrity of our hereditary material is constantly challenged by both endogenous agents formed during normal cellular metabolism and by various exogenous factors, like UV-light and chemicals found everywhere in the environment. Damage in DNA can block genome replication, which can lead to genomic instability and death of the cell. To overcome these blocks, organisms have evolved DNA damage tolerance mechanisms that allow completion of DNA replication in the presence of damage. One of them is translesion DNA synthesis (TLS), which is mediated by specialized TLS DNA polymerases that are able replicate over the replication-blocking DNA damage. Information encoded by a damaged nucleotide is usually inaccurate, therefore TLS is inherently error-prone process. As such, TLS polymerases not only protect cells against DNA damage, but also represent a potential source of mutations – a material for evolution, but also a cause of disease in humans. In bacteria, genetic diversity generated by TLS polymerases is critical for the acquisition of antibiotic resistance. Therefore, a lot of research is now undertaken to use TLS polymerases as a target for cancer or antimicrobial treatment. In the present work, I describe the role of TLS polymerases in two representatives of Pseudomonas, important human pathogen Pseudomonas aeruginosa and soil bacterium Pseudomonas putida. These organisms possess three TLS polymerases: Pol II, Pol IV and ImuABC. The results of our work revealed that in P. putida TLS polymerases might leave mutagenic fingerprints in the genome during normal growth of bacteria, suggesting their potential role in bypass past endogenously formed DNA damage. Moreover, Pol II and Pol IV might be involved in DNA replication in the absence of replicative DNA polymerase I. Both Pol IV and ImuABC in Pseudomonads are involved in DNA alkylation damage tolerance. Replication across alkylation damage by ImuABC is highly mutagenic, Pol IV, on the contrary, performs very accurate bypass. One of the important findings of my study was that simple switch in growth temperature of bacteria changed the cell’s strategy to deal with alkylation DNA damage and role of TLS in it.
Description
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Keywords
mutations, DNA damage, DNA polymerase, DNA repair