Accurate genome maintenance is critical to prevent the accumulation of genomic and epigenomic aberrations, which can result in defective cell function and disease. DNA double-strand breaks (DSBs) are perhaps the most severe threat to genome integrity and are often the result of replication errors. If unrepaired, DSBs cause cell cycle arrest, cell death or chromosomal translocations - a known driver of malignant transformation. DSB repair is equally essential for tumor growth, e.g. by counteracting replication stress resulting from excessive tumor cell division. As a result, tumor cells frequently become addicted to a specific repair pathway, particularly when alternative pathways are defective. A prominent example for the latter are tumors with mutations in the Breast Cancer (BRCA) tumor suppressor genes, which are required for DSB repair via homologous recombination (HR). BRCA1 mutations are synthetically lethal with defects in alternative end joining, a backup repair pathway often induced in BRCA1-deficient tumors. The targeted manipulation of repair pathways is thus emerging as an important means not only to prevent malignant transformation but also to interfere with tumor growth. The resolution of DSBs generally involves one of three major DSB repair pathways - HR, non-homologous end joining (NHEJ) or alternative NHEJ (alt-NHEJ). Access and choice of the appropriate repair factors is in turn tightly controlled by alterations in the DSB-proximal chromatin environment. In addition, multiple reports have described the accumulation of RNA processing enzymes as well as RNA and/or RNA:DNA hybrid structures at DSBs. Underlining the importance of RNA for DSB repair, RNase treatment was shown to impair successful DSB resolution. Moreover, RNA has recently been reported to be transcribed from DSB ends to facilitate downstream repair factor recruitment. However, in contrast to chromatin, little is known about how RNA modulates the repair process. Recent work points to RNA modifications as important contributing factors, as UV-induced single-stranded DNA lesions cause increased levels of the modification N6-methyladenosine (m6A) in RNA. m6A is present in numerous coding and non-coding RNA species, and its addition or removal through the activity of methylases (writers) and demethylases (erasers) can alter RNA structure and/or its interaction with proteins as well as nucleic acids. Supporting a role for m6A metabolism in the cellular response to DNA damage beyond UV, our preliminary data show that (i) nuclear m6A-modified RNA accumulates at laser- as well as endonuclease-induced DSBs and upon ionizing irradiation (IR), (ii) IR-induced m6A accumulation is at least in part dependent on the predominant m6A RNA methyltransferase METTL3, and (iii) loss of METTL3 causes increased DNA break-associated chromosomal aberrations and reduced efficiency of non-homologous end joining (NHEJ). Of relevance for cancer genome maintenance, METTL3 depletion significantly impaired the recruitment of 53BP1, an NHEJ factor and BRCA1 antagonist, which was found to account for PARP inhibitor sensitivity of BRCA1 mutant cancers. We, thus, hypothesize that m6A-modified RNA controls the temporal and spatial control of repair factor function in response to DSBs. Of note, disruption of the m6A pathway by deregulation of METTL3 as well as m6A demethylases, such as ALKBH5 and FTO, has been linked to breast cancer progression. However, the molecular basis for these observations remains largely unknown. In this project, which is a collaborative effort with Dr. Pedro Batista at NCI, we combine two highly cancer-relevant research areas, DNA repair and RNA metabolism, to investigate a previously unexplored aspect of genome maintenance: the control of DSB repair by RNA and its most common post-transcriptional modification, m6A.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011814-01
Application #
9780091
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Project End
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Budget End
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
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Sebastian, Robin; Oberdoerffer, Philipp (2017) Transcription-associated events affecting genomic integrity. Philos Trans R Soc Lond B Biol Sci 372: