Sjoerd Wanrooij - Assistant Professor
Molecular mechanism of DNA repair and replication in human mitochondria
Human mitochondrial DNA (mtDNA) encodes for genes that are essential for proper energy production (Figure 1). Loss of mtDNA function results in a broad range of genetic human disorders, and it is increasingly recognized as a significant factor in common disorders such as neurodegeneration. Our group investigates detailed molecular mechanisms of mitochondrial DNA replication and repair in order to better understand the underlying causes of such disease states.
Mitochondrial DNA is replicated by an unique enzymatic machinery that is completely distinct from the DNA replication machinery that maintains the nuclear genome (Figure 2). The basal mtDNA replication machinery can be reconstituted in vitro using highly purified, recombinant proteins and a DNA substrate. Even though many of the key components present at the mammalian mtDNA replication fork have been identified, there is still an ongoing scientific debate about the exact mechanisms of mtDNA replication.
During years of research in DNA repair, the mitochondrial repair pathways have been very much neglected. This is due to the general long-standing misconception that mitochondria do not contain DNA repair activity, while at the same time the repair of nuclear DNA has proven to be of utmost importance in human disease (e.g. cancer). Only recent discoveries have shown that there are extensive mitochondrial DNA repair pathways and that this process is implicated in human ageing-related diseases.
The focus of our group is on the processes of mitochondrial DNA replication and repair. Much of the research consists of in vitro biochemical experiments in combination with cell biological approaches to verify any in vivo relevance of the observations.
Natalie Al-Furoukh, Post Doc
Rafil Khairullin, Post Doc
Gorazd Stojkovic, Post Doc
Mara Doimo, Post Doc
Annika Pfeiffer, Post Doc
Xin Zhou, Post Doc
Sonja Stenmark, Senior research engineer
Josefin Forslund, PhD-student
Erika Larsson, Undergraduate student
Stojkovič G, Makarova AV, Wanrooij PH, Forslund J, Burgers PM, Wanrooij S (2016): Oxidative DNA damage stalls the human mitochondrial replisome. Sci Rep. 6:28942. doi: 10.1038/srep28942.
Al-Furoukh N, Ianni A, Nolte H, Hölper S, Krüger M, Wanrooij S and Braun T (2015): ClpX stimulates the mitochondrial unfolded protein response (UPR(mt)) in mammalian cells. Biochim Biophys Acta 1853:2580-91
Fusté JM, Shi Y, Wanrooij S, Zhu X, Jemt E, Persson Ö, Sabouri N, Gustafsson CM and Falkenberg M (2014): In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of replication. PLoS Genetics 10(12):e1004832
Al-Furoukh N, Goffart S, Sibor M, Wanrooij S and Braun T (2013): NOA1 couples G-quadruplex binding and GTP hydrolysis. Biochim Biophys Acta 1833: 2933-2942.
Shi Y, Dierckx A, Wanrooij PH, Wanrooij S, Larsson NG, Wilhelmsson LM, Falkenberg M and Gustafsson CM (2012): Mammalian transcription factor A is a core component of the mitochondrial transcription machinery. Proc Natl Acad Sci USA 109: 16510-16515.
Wanrooij S, Miralles Fuste J, Stewart JB, Wanrooij PH, Samuelsson T, Larsson NG, Gustafsson CM and Falkenberg M (2012): In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication. EMBO Rep 13: 1130-1137.
Atanassova N, Fuste JM, Wanrooij S, Macao B, Goffart S, Bäckström S, Farge G, Khvorostov I, Larsson NG Spelbrink JN and Falkenberg M (2011): Sequence-specific stalling of DNA polymerase gamma and the effects of mutations causing progressive ophthalmoplegia. Hum Mol Genet 20: 1212-1223.
Wanrooij S, Falkenberg M (2010): The human mitochondrial replication fork in health and disease. Biochim Biophys Acta 1797: 1378-1388.
Wanrooij S, Fuste JM, Jemt E, Granycome EG, Cluett TJ, Shi Y, Atanassova N, Holt IJ, Gustafsson CM and Falkenberg M (2010): Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication. Mol Cell 37: 67-78.
Goffart S, Cooper HM, Tyynismaa H, Wanrooij S, Suomalainen A and Spelbrink JN (2009): Twinkle mutations associated with autosomal dominant progressive external opthalmoplegia lead to impaired helicase function and in vivo mtDNA replication stalling. Hum Mol Genet 18: 328-40.
Wanrooij S, Fuste JM, Farge G, Shi Y, Gustafsson CM and Falkenberg M (2008): Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro. Proc Natl Acad Sci USA 105: 11122-11127. Citations 39.
Krishnan KJ, Reeve AK, Samuels DC, Chinnery PF, Blackwood JK, Taylor RW, Wanrooij S, Spelbrink JN, Lightowlers RN and Turnbull DM (2008): What causes mitochondrial DNA deletions in human cells? Nat Genet 40: 275-279.
Hyvärinen A, Pohjoismäki JLO, Reyes A, Wanrooij S, Yasukawa T, Karhunen P, Spelbrink JN, Holt IJ (2007): The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA. Nucleic Acids Res 35: 6458-6474.
Wanrooij S, Goffart S, Pohjoismäki JLO, Yasukawa T and Spelbrink JN (2007): Expression of catalytic mutants of the mtDNA helicase and polymerase POLG causes distinct replication stalling phenotypes. Nucleic Acids Res 35: 3238-3251
Pohjoismäki JL, Wanrooij S, Hyvärinen AK, Goffart S, Holt IJ, Spelbrink JN and Jacobs HT (2006): Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells. Nucleic Acids Res 34: 5815-5828.
Tyynismaa H, Mjosund KP, Wanrooij S, Lappalainen I, Ylikallio E, Jalanko A, Spelbrink JN, Paetau A and Suomalainen A (2005): Mutant mitochondrial helicase Twinkle causes multiple mtDNA deletions and a late-onset mitochondrial disease in mice. Proc Natl Acad Sci U S A 102: 17687-17692.
Wanrooij S, Luoma P, van Goethem G, van Broeckhoven C, Suomalainen A and Spelbrink JN (2004): Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA. Nucleic Acids Res 32: 3053-3064.
Spelbrink JN, Li FY, Tiranti V, Nikali K, Yuan QP, Tariq M, Wanrooij S, Garrido N, Comi G, Morandi L, Santoro L, Toscano A, Fabrizi GM, Croxen R, Beeson D, Poulton J, Suomalainen A, Jacobs HT, Zeviani M and Larsson C (2001) Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet 28: 223-231.