The retrotransposon R2 maintains Drosophila ribosomal DNA repeats

Ribosomal RNAs (rRNAs) account for 80-90% of all transcripts in eukaryotic cells. To meet this demand, the ribosomal DNA (rDNA) gene that codes for rRNA is tandemly repeated hundreds of times, comprising rDNA loci on eukaryotic chromosomes. This repetitiveness imposes a challenge to maintaining sufficient copy number due to spontaneous intra-chromatid recombination between repetitive units causing copy number loss. The progressive shrinking of rDNA loci from generation to generation could lead to extinction of the lineage, yet the mechanism(s) to counteract spontaneous copy number loss remained unclear. Here, we show that the rDNA-specific retrotransposon R2 is essential for rDNA copy number (CN) maintenance in the Drosophila male germline, despite the perceived disruptive nature of transposable elements. Depletion of R2 led to defective rDNA CN maintenance in multiple contexts, causing a decline in fecundity over generations and eventual extinction of the lineage. Our data suggests that DNA double strand breaks generated by R2 is the initiating event of rDNA CN expansion, stimulating the repair processes proposed to underlie rDNA CN expansion. This study reveals that retrotransposons can provide a benefit to their hosts, contrary to their reputation as genomic parasitic, which may contribute to their widespread success throughout taxa.

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Recent advances in the nucleolar responses to DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa713 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9449-9461

Author(s):

Lea Milling Korsholm ◽

Zita Gál ◽

Blanca Nieto ◽

Oliver Quevedo ◽

Stavroula Boukoura ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Ribosomal Dna ◽

Repetitive Sequences ◽

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

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The Shu complex, which contains Rad51 paralogues, promotes DNA repair through inhibition of the Srs2 anti-recombinase

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0691 ◽

2011 ◽

Vol 22 (9) ◽

pp. 1599-1607 ◽

Cited By ~ 55

Author(s):

Kara A. Bernstein ◽

Robert J.D. Reid ◽

Ivana Sunjevaric ◽

Kimberly Demuth ◽

Rebecca C. Burgess ◽

...

Keyword(s):

Copy Number ◽

Dna Helicase ◽

Double Strand Breaks ◽

General Function ◽

Rdna Transcription ◽

Strand Breaks ◽

Important Regulator ◽

Error Prone Repair ◽

Rdna Copy ◽

Rdna Copy Number

The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. We discovered a link to ribosomal DNA (rDNA) recombination when we found an interaction between one member of the Shu complex, SHU1, and UAF30, a component of the upstream activating factor complex (UAF), which regulates rDNA transcription. In the absence of Uaf30, rDNA copy number increases, and this increase depends on several functional subunits of the Shu complex. Furthermore, in the absence of Uaf30, we find that Shu1 and Srs2, an anti-recombinase DNA helicase with which the Shu complex physically interacts, act in the same pathway regulating rDNA recombination. In addition, Shu1 modulates Srs2 recruitment to both induced and spontaneous foci correlating with a decrease in Rad51 foci, demonstrating that the Shu complex is an important regulator of Srs2 activity. Last, we show that Shu1 regulation of Srs2 to double-strand breaks is not restricted to the rDNA, indicating a more general function for the Shu complex in the regulation of Srs2. We propose that the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2.

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Hedgehog signaling enables repair of ribosomal DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa733 ◽

2020 ◽

Vol 48 (18) ◽

pp. 10342-10352

Author(s):

Tshering D Lama-Sherpa ◽

Victor T G Lin ◽

Brandon J Metge ◽

Shannon E Weeks ◽

Dongquan Chen ◽

...

Keyword(s):

Cancer Cells ◽

Ribosomal Dna ◽

Hedgehog Signaling ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Rdna Sequences ◽

Polymerase I

Abstract Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.

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Transgenerational dynamics of rDNA copy number in Drosophila male germline stem cells

10.1101/199679 ◽

2017 ◽

Author(s):

Kevin L. Lu ◽

Jonathan O. Nelson ◽

Natalie Warsinger-Pepe ◽

Yukiko M. Yamashita

Keyword(s):

Copy Number ◽

Replicative Senescence ◽

Yeast Cells ◽

Rrna Genes ◽

Germline Stem Cell ◽

Genetic Elements ◽

Male Germline ◽

Rdna Loci ◽

Rdna Copy ◽

Rdna Copy Number

AbstractrDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. In this study, we investigate the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. Our study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in animals that inherit reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, we propose that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

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Transgenerational dynamics of rDNA copy number in Drosophila male germline stem cells

eLife ◽

10.7554/elife.32421 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 20

Author(s):

Kevin L Lu ◽

Jonathan O Nelson ◽

George J Watase ◽

Natalie Warsinger-Pepe ◽

Yukiko M Yamashita

Keyword(s):

Copy Number ◽

Replicative Senescence ◽

Yeast Cells ◽

Rrna Genes ◽

Germline Stem Cell ◽

Genetic Elements ◽

Male Germline ◽

Rdna Loci ◽

Rdna Copy ◽

Rdna Copy Number

rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. In this study, we investigate the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. Our study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in young animals that inherited reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, we propose that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

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Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5′-Phosphorylated DNA Double-Strand Breaks by Replication Runoff

Molecular and Cellular Biology ◽

10.1128/mcb.20.11.3977-3987.2000 ◽

2000 ◽

Vol 20 (11) ◽

pp. 3977-3987 ◽

Cited By ~ 223

Author(s):

Dirk Strumberg ◽

André A. Pilon ◽

Melanie Smith ◽

Robert Hickey ◽

Linda Malkas ◽

...

Keyword(s):

Dna Polymerase ◽

Ribosomal Dna ◽

Topoisomerase I ◽

Human Colon ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dna Damages ◽

Strand Breaks ◽

Leading Strand

ABSTRACT Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3′ DNA ends are extended by DNA polymerase in vivo closely to the 5′ ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5′ ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5′ kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.

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Repair pathway choice for double-strand breaks

Essays in Biochemistry ◽

10.1042/ebc20200007 ◽

2020 ◽

Vol 64 (5) ◽

pp. 765-777 ◽

Cited By ~ 2

Author(s):

Yixi Xu ◽

Dongyi Xu

Keyword(s):

Cell Cycle ◽

Double Strand Breaks ◽

Homologous Region ◽

Gene Repair ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

Environmental Radiation ◽

Strand Breaks ◽

Dna End Resection ◽

End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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