A moonlighting metabolic protein influences repair at DNA double-stranded breaks.

Catalytically active proteins with divergent dual functions are often described as 'moonlighting'. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair

Roles of Homologous Recombination in Processing DNA Lesions

8 9 Scope of the Thesis Scope of the Thesis The abundance of DNA damaging agents poses a constant threat for genome stability. Therefore, cells have evolved multiple mechanisms to repair their DNA. The variety in possible DNA lesions that can occur require specified repair mechanism with the ability to remove particular types of lesions. For example, nucleotide excision repair removes lesions generated by UV-light whereas homologous recombination repairs DNA double-stranded breaks induced by ionising radiation. However, the same type of DNA lesion can b

Stemness factor Sall4 is required for DNA damage response in embryonic stem cells.

Mouse embryonic stem cells (ESCs) are genetically more stable than somatic cells, thereby preventing the passage of genomic abnormalities to their derivatives including germ cells. The underlying mechanisms, however, remain largely unclear. In this paper, we show that the stemness factor Sall4 is required for activating the critical Ataxia Telangiectasia Mutated (ATM)-dependent cellular responses to DNA double-stranded breaks (DSBs) in mouse ESCs and confer their resistance to DSB-induced cytotoxicity. Sall4 is rapidly mobilized to the sites of DSBs after DNA damage. Furthermore, Sall4 interacts with Rad50 and stabilizes the Mre11-Rad50-Nbs1 complex for the efficient recruitment and activation of ATM. Sall4 also interacts with Baf60a, a member of the SWI/SNF (switch/sucrose nonfermentable) ATP-dependent chromatin-remodeling complex, which is responsible for recruiting Sall4 to the site of DNA DSB damage. Our findings provide novel mechanisms to coordinate stemness of ESCs with DNA damage response, ensuring genomic stability during the expansion of ESCs

BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ

SummaryWe showed in this study that cells deficient of the BRCA1-associated BACH1 helicase, also known as BRIP1, failed to elicit homologous recombination (HR) after DNA double-stranded breaks (DSBs). BACH1-deficient cells were also sensitive to mitomycin C (MMC) and underwent MMC-induced chromosome instability. Moreover, we identified a homozygous nonsense mutation in BACH1 in a FA-J patient-derived cell line and could not detect BACH1 protein in this cell line. Expression of wild-type BACH1 in this cell line reduced the accumulation of cells at G2/M phases following exposure to DNA crosslinkers, a characteristic of Fanconi anemia (FA) cells. These results support the conclusion that BACH1 is FANCJ

Analysis of the role of Sgs1p in repair of double-strand DNA breaks in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

Mis-repair of DNA double-stranded breaks (DSB) can result in genomic rearrangements and cancer. Sgs1p is a helicase that unwinds DNA for DSB repair. To better understand its role in break repair, I analyzed the frequency of various types of DSB repairs in yeast cells with the mutation sgs1-FD. Given this mutation disrupts an interaction between Sgs1p and a critical protein, Rad51p, I hypothesized mutant cells would have more error-prone repair than high fidelity canonical BIR repair. Genome sequence and chromosome size were examined in sgs1-FD yeast cells that underwent DSB repair. No difference was found in the frequency of error-prone repair, indicating the mutation did not affect the repair process

Rapid Integration of Multi-copy Transgenes Using Optogenetic Mutagenesis in Caenorhabditis elegans.

Stably transmitted transgenes are indispensable for labeling cellular components and manipulating cellular functions. In Caenorhabditis elegans, transgenes are generally generated as inheritable multi-copy extrachromosomal arrays, which can be stabilized in the genome through a mutagenesis-mediated integration process. Standard methods to integrate extrachromosomal arrays primarily use protocols involving ultraviolet light plus trimethylpsoralen or gamma- or X-ray irradiation, which are laborious and time-consuming. Here, we describe a one-step integration method, following germline-mutagenesis induced by mini Singlet Oxygen Generator (miniSOG). Upon blue light treatment, miniSOG tagged to histone (Histone-miniSOG) generates reactive oxygen species (ROS) and induces heritable mutations, including DNA double-stranded breaks. We demonstrate that we can bypass the need to first establish extrachromosomal transgenic lines by coupling microinjection of desired plasmids with blue light illumination on Histone-miniSOG worms to obtain integrants in the F3 progeny. We consistently obtained more than one integrant from 12 injected animals in two weeks. This optogenetic approach significantly reduces the amount of time and labor for transgene integration. Moreover, it enables to generate stably expressed transgenes that cause toxicity in animal growth

Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids

Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability

RuvAB Acts at Arrested Replication Forks

AbstractReplication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage

DNA Double Strand Breaks and Chromosomal Translocations Induced by DNA Topoisomerase II

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.Spanish Ministry of Science and Universities BFU2016-76446-P, RYC-2014-1666