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Translesion DNA polymerases are required for spontaneous deletion formation in Salmonella typhimurium
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Dan Andersson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Dan Andersson)
2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 25, 10248-10253 p.Article in journal (Refereed) Published
Abstract [en]

How spontaneous deletions form in bacteria is still a partly unresolved problem. Here we show that deletion formation in S. typhimurium requries the presence of functional translesion polymerases. First, in wild type bacteria, removal of the known translesion DNA polymerases: PolII (polB), PolIV (dinB), PolV (umuDC) and the PolV homologue SamAB (samAB) resulted in a 10-fold decrease in the deletion rate, indicating that 90% of all spontaneous deletions require these polymerases for their formation. Second, overexpression of these polymerases by de-repression of the DNA damage-inducible LexA regulon caused a 25-fold increase in deletion rate that depended on the presence of functional translesion polymerases. Third, overexpression of the polymerases PolII and PolIV from a plasmid increased the deletion rate 12- to 30-fold respectively. Last, in a recBC- mutant where dsDNA ends are stabilized due to the lack of the end-processing nuclease RecBC, the deletion rate was increased 20-fold. This increase depended on the translesion polymerases. In lexA(def) mutant cells with constitutive SOS-expression, a 10-fold increase in DNA breaks was observed. Inactivation of all 4 translesion polymerases in the lexA(def) mutant reduced the deletion rate 250-fold without any concomitant reduction in the amount of DNA breaks. Mutational inactivation of 3 endonucleases under LexA control, reduced the number of DNA breaks to the wild-type level in a lexA(def) mutant with a concomitant 50-fold reduction in deletion rate. These findings suggest that the translesion polymerases are not involved in forming the DNA breaks, but that they require them to stimulate deletion formation.

Place, publisher, year, edition, pages
2009. Vol. 106, no 25, 10248-10253 p.
Keyword [en]
bacteria, DNA homology, gene loss, RecA protein
National Category
Medical and Health Sciences
Research subject
Evolutionary Genetics
Identifiers
URN: urn:nbn:se:uu:diva-111424DOI: 10.1073/pnas.0904389106ISI: 000267292200034OAI: oai:DiVA.org:uu-111424DiVA: diva2:281150
Available from: 2009-12-14 Created: 2009-12-14 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Dynamics of the Bacterial Genome: Rates and Mechanisms of Mutation
Open this publication in new window or tab >>Dynamics of the Bacterial Genome: Rates and Mechanisms of Mutation
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacterial chromosomes are highly dynamic, continuously changing with respect to gene content and size via a number of processes, including deletions that result in gene loss. How deletions form and at what rates has been the focus of this thesis.

In paper II we investigated how chromosomal location affects chromosomal deletion rates in S. typhimurium. Deletion rates varied more than 100-fold between different chromosomal locations and some large deletions significantly increased the exponential growth rate of the cells. Our results suggest that the chromosome is heterogeneous with respect to deletion rates and that deletions may be genetically fixed as a consequence of natural selection rather than by drift or mutational biases.

In paper I we examined in a laboratory setting how rapidly reductive evolution, i.e. gene loss, could occur. Using a serial passage approach, we showed that extensive genome reduction potentially could occur on a very short evolutionary time scale. For most deletions we observed little or no homology at the deletion endpoints, indicating that spontaneous deletions often form through a RecA independent process.

In paper III we examined further how large spontaneous deletions form and, unexpectedly, showed that 90% of all spontaneous chromosomal deletions required error-prone translesion DNA polymerases for their formation. We propose that the translesion polymerases stimulate deletion formation by allowing extension of misaligned single-strand DNA ends.

In paper IV we investigated how the translesion DNA polymerase Pol IV, RpoS and different types of stresses affect mutation rates in bacteria. Derepression of the LexA regulon caused a small to moderate increase in mutation rates that was fully dependent on functional endonucleases but only partly dependent on translesion DNA polymerases. RpoS levels and growth stresses had only minor effects on mutation rates. Thus, mutation rates appear very robust and are only weakly affected by growth conditions and induction of translesion polymerases and RpoS.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 56 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 509
Keyword
bacteria, bacterial evolution, genome reduction, gene loss, serial passage, DNA homology, tranlesion DNA polymerase, stress
National Category
Microbiology in the medical area Microbiology in the medical area
Research subject
Evolutionary Genetics; Microbiology
Identifiers
urn:nbn:se:uu:diva-111428 (URN)978-91-554-7687-8 (ISBN)
Public defence
2010-02-05, C10:305, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2010-01-14 Created: 2009-12-14 Last updated: 2010-01-15

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Koskiniemi, SannaAndersson, Dan I

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