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- Kreisel, Katrin, 1991, et al.
(författare)
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DNA polymerase η contributes to genome-wide lagging strand synthesis.
- 2019
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Ingår i: Nucleic acids research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 47:5, s. 2425-2435
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Tidskriftsartikel (refereegranskat)abstract
- DNA polymerase η (pol η) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol ηalso has other cellular roles. Here, we present evidence that pol η competes with DNA polymerases α and δfor the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol η,which contains a PCNA-Interacting Protein motif is required for pol ηto function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol η and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.
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| 2. |
- Kreisel, Katrin, 1991
(författare)
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Ribonucleotides in DNA - Application in genome-wide DNA polymerase tracking and physiological role in eukaryotes
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The genetic code in the eukaryotic cell is stored in the form of DNA, which is more resistant to hydrolysis than RNA. Replication fidelity and DNA repair mechanisms are in place to ensure genomic integrity to preserve the information encoded. Despite DNA polymerases’ discrimination against ribonucleotides, they are frequently incorporated into DNA and even in the presence of efficient ribonucleotide removal pathways, ribonucleotides may remain stably incorporated in the DNA. Ribonucleotides can be used as a marker of DNA replication enzymology by using HydEn-seq, a next-generation sequencing technique for the genome-wide mapping of ribonucleotides. I aimed to elucidate the activities of the specialized translesion synthesis DNA polymerase η in yeast. By using a steric gate variant that incorporates more ribonucleotides and by tracking those ribonucleotides, I determined a lagging strand preference dependent on its C-terminus in Paper I. The findings suggest a possible extension of the ‘division of labor’ among replicative polymerases to the specialized polymerases. Moreover, I was interested in the physiological role of incorporated ribonucleotides and used an extension of the HydEn-seq method outlined in Paper II, to map and quantitate ribonucleotides simultaneously. By investigating ribonucleotide incorporation into mouse mitochondrial DNA (mtDNA) in Paper III, we found that ribonucleotides are acquired mostly up until adulthood and are not connected to age-related mtDNA instability, suggesting relatively good tolerance of incorporated ribonucleotides in mtDNA. To gain a more comprehensive view on incorporated ribonucleotides in the DNA of mammals, I mapped and quantitated incorporated ribonucleotides in nuclear DNA (nDNA) and mtDNA from murine blood, bone marrow, brain, heart, kidney, liver, lung, muscle and spleen in Paper IV. I found tissue-dependent variations in the number and the identity of incorporated ribonucleotides and marked differences between nDNA and mtDNA. The ribonucleotide distribution in both types of DNA was non-random and in nDNA affected by the proximity of genomic features, which in most cases increased the number of embedded ribonucleotides locally as compared to random positions in the nDNA. The thesis extends the knowledge of DNA polymerase η’s activity and the physiological role that incorporated ribonucleotides play in DNA. This more detailed characterization of the incorporated ribonucleotides genome-wide is a basic requirement for the understanding of diseases associated with genome instability, such as certain types of cancers or Aicardi-Goutières syndrome.
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| 3. |
- Kreisel, Katrin, 1991, et al.
(författare)
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Simultaneous mapping and quantitation of ribonucleotides in human mitochondrial DNA
- 2017
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; 2017
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Tidskriftsartikel (refereegranskat)abstract
- © 2017. Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5´-OH ends or 5´-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5´-end processing, DNA damage, and DNA repair.
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