Change search
ReferencesLink to record
Permanent link

Direct link
Transcription Associated Recombination in Mammalian Cells
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is increasing evidence that the movement of the transcription machinery through DNA has profound effects on the genomic stability. One such example is a phenomenon known as Transcription Associated Recombination (TAR). Transcription enhances recombination levels to a high degree in all organisms studied, from bacteria to mammals. The underlying causes of the high recombination levels observed are unknown, as are the rationale for the rather riskyhazardous recombination event in this context. Recombination is not a risk-free event; there is e.g. the chancerisk for of loss of heterozygozity, which may eventually lead to tumour formation. So, why is TAR so ubiquitous? This thesis deals with the factors inducing TAR, trying to elucidate the mechanisms catalyzing this event. The proteins involved in executing TAR are unknown in mammals, and one of the aims of this thesis havehas been to investigate the role of well-known DNA repair proteins in TAR. In order to do so, cell lines deficient in crucial DNA repair proteins were stably transfected with a novel recombination construct. Transcription can be controlled over this recombination construct, enabling the detection of transcription associated recombination. We found that TAR is dependent on replication and that inhibition of transcription elongation had no further effect on TAR levels in our system. Further, we found that TAR employs a recombination pathway mechanistically separate from the recombination pathway induced by DNA double strand breaks. This pathway is dependent on BRCA2, a protein required for homologous recombination, but independent of the RAD51 paralog XRCC2. In subsequent studies, we found that the XPD subunit of the combined transcription and repair factor TFIIH is required for TAR, but is dispensable for DNA DSB repair by HR. We went on to investigate the connection between HR repair of UV damages and transcription and found that repair of UV damages requires transcription, but not via the transcription-coupled repair pathway. In conclusion, we found that TAR operates by a recombination pathway separate from DNA double strand break induced recombination. We found a connection with stalled replication, and revealed several of the proteins required for TAR in mammals.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University , 2010. , 62 p.
National Category
Biological Sciences
Research subject
Molecular Genetics
URN: urn:nbn:se:su:diva-38931ISBN: 978-91-7447-079-6OAI: diva2:317581
Public defence
2010-06-03, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.Available from: 2010-05-11 Created: 2010-05-04 Last updated: 2011-02-15Bibliographically approved
List of papers
1. Transcription-associated recombination is dependent on replication in Mammalian cells
Open this publication in new window or tab >>Transcription-associated recombination is dependent on replication in Mammalian cells
2008 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 1, 154-64 p.Article in journal (Refereed) Published
Abstract [en]

Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.

Animals, Cell Cycle, Cell Line, Cell Separation, Cricetinae, Cricetulus, DNA/*genetics, DNA Damage/genetics, DNA Replication/*genetics, Recombination; Genetic/*genetics, Signal Transduction, Transcription; Genetic/*genetics
National Category
Cell and Molecular Biology
urn:nbn:se:su:diva-11830 (URN)10.1128/MCB.00816-07 (DOI)000251925300013 ()17967877 (PubMedID)
Available from: 2008-01-14 Created: 2008-01-14 Last updated: 2010-05-07Bibliographically approved
2. Transcription-associated recombination is independent of XRCC2 and mechanistically separate from homology-directed DNA double-strand break repair
Open this publication in new window or tab >>Transcription-associated recombination is independent of XRCC2 and mechanistically separate from homology-directed DNA double-strand break repair
2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 2, 405-412 p.Article in journal (Refereed) Published
Abstract [en]

It has previously been shown that transcription greatly enhances recombination in mammalian cells. However, the proteins involved in catalysing this process and the recombination pathways involved in transcription-associated recombination (TAR) are still unknown. It is well established that both the BRCA2 protein and the RAD51 paralog protein XRCC2 are required for homologous recombination. Here, we show that the BRCA2 protein is also required for TAR, while the XRCC2 protein is not involved. Expression of the XRCC2 gene in XRCC2 mutated irs1 cells restores the defect in homologous recombination repair of an I-SceI-induced DNA double-strand break, while TAR is unaffected. Interestingly, the XRCC2-deficient irs1 cells are also proficient in recombination induced at slowed replication forks, suggesting that TAR is mechanistically linked with this recombination pathway. In conclusion, we show that TAR depends on BRCA2 but is independent of XRCC2, and that this recombination pathway is separate from that used to repair a two-ended DNA double-strand break.

National Category
Microbiology in the medical area
urn:nbn:se:su:diva-32012 (URN)10.1093/nar/gkn971 (DOI)000262963400017 ()19043071 (PubMedID)
Available from: 2009-12-02 Created: 2009-12-02 Last updated: 2010-05-07Bibliographically approved
3. The XPD subunit of TFIIH is required for transcription-associated but not DNA double-strand break-induced recombination in mammalian cells
Open this publication in new window or tab >>The XPD subunit of TFIIH is required for transcription-associated but not DNA double-strand break-induced recombination in mammalian cells
2010 (English)In: Mutagenesis, ISSN 0267-8357, E-ISSN 1464-3804, Vol. 25, no 6, 623-629 p.Article in journal (Other academic) Published
Abstract [en]

Mutations in the XPD gene can give rise to three phenotypically distinct disorders: xeroderma pigmentosum (XP),  trichothiodystrophy (TTD) or combined XP and Cockayne syndrome (CS) (XP/CS). The role of XPD in nucleotide excision repair explains the increased risk of skin cancer in XP patients, but not all the clinical phenotypes found in XP/CS or TTD patients. Here, we describe that the XPD defective UV5 cell line is impaired in transcription-associated recombination (TAR), which can be reverted by the introduction of the wild type XPD gene expressed from a vector. UV5 cells are defective in TAR, despite having intact transcription and homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Interestingly, we find reduced spontaneous HR in XPD defective cells, suggesting that transcription underlie a portion of spontaneous HR events. We also report that transcription-coupled repair (TCR) defective CSB cells, have a defect in TAR, but not in DSB-induced HR. However, the TAR defect may be associated with a general transcription defect in CSB deficient cells.  In conclusion, we show a novel role for the XPD protein in TAR, linking TAR with TCR.

National Category
Natural Sciences
urn:nbn:se:su:diva-39035 (URN)10.1093/mutage/geq054 (DOI)
Available from: 2010-05-06 Created: 2010-05-06 Last updated: 2011-01-20Bibliographically approved
4. Transcription-associated UV-induced DNA damage triggers futile homologous recombination repair in mammalian cells
Open this publication in new window or tab >>Transcription-associated UV-induced DNA damage triggers futile homologous recombination repair in mammalian cells
Show others...
(English)Manuscript (preprint) (Other academic)
urn:nbn:se:su:diva-39036 (URN)
Available from: 2010-05-06 Created: 2010-05-06 Last updated: 2010-05-07Bibliographically approved

Open Access in DiVA

fulltext(644 kB)467 downloads
File information
File name FULLTEXT01.pdfFile size 644 kBChecksum SHA-512
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Savolainen, Linda
By organisation
Department of Genetics, Microbiology and Toxicology
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 467 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 211 hits
ReferencesLink to record
Permanent link

Direct link