Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability.

Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.

DNA repair and the control of DNA methylation.

The successful establishment and stable maintenance of cell identity are critical for organismal development and tissue homeostasis. Cell identity is provided by epigenetic mechanisms that facilitate a selective readout of the genome. Operating at the level of chromatin, they establish defined gene expression programs during cell differentiation. Among the epigenetic modifications in mammalian chromatin, the 5'-methylation of cytosine in CpG dinucleotides is unique in that it affects the DNA rather than histones and the biochemistry of the DNA methylating enzymes offers a mechanistic explanation for stable inheritance. Yet, DNA methylation states appear to be more dynamic and their maintenance more complex than existing models predict. Also, methylation patterns are by far not always faithfully inherited, as best exemplified by human cancers. Often, these show widespread hypo- or hypermethylation across their genomes, reflecting an underlying epigenetic instability that may have contributed to carcinogenesis. The phenotype of unstable methylation in cancer illustrates the importance of quality control in the DNA methylation system and implies the existence of proof-reading mechanisms that enforce fidelity to DNA methylation in healthy tissue. Fidelity seems particularly important in islands of unmethylated CpG-rich sequences where an accurate maintenance of un- or differentially methylated states is critical for stable expression of nearby genes. Methylation proof-reading in such sequences requires a system capable of recognition and active demethylation of erroneously methylated CpGs. Active demethylation of 5-methylcytosine has been known to occur for long, but the underlying mechanisms have remained enigmatic and controversial. However, recent progress in this direction substantiates a role of DNA repair in such processes. This review will address general aspects of cytosine methylation stability in mammalian DNA and explore a putative role of DNA repair in methylation control.

DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier.

DNA double-strand breaks (DSB) were shown to occur at the replication fork barrier in the ribosomal DNA of Saccharomyces cerevisiae using 2D-gel electrophoresis. Their origin, nature and magnitude, however, have remained elusive. We quantified these DSBs and show that a surprising 14% of replicating ribosomal DNA molecules are broken at the replication fork barrier in replicating wild-type cells. This translates into an estimated steady-state level of 7-10 DSBs per cell during S-phase. Importantly, breaks detectable in wild-type and sgs1 mutant cells differ from each other in terms of origin and repair. Breaks in wild-type, which were previously reported as DSBs, are likely an artefactual consequence of nicks nearby the rRFB. Sgs1 deficient cells, in which replication fork stability is compromised, reveal a class of DSBs that are detectable only in the presence of functional Dnl4. Under these conditions, Dnl4 also limits the formation of extrachromosomal ribosomal DNA circles. Consistently, dnl4 cells displayed altered fork structures at the replication fork barrier, leading us to propose an as yet unrecognized role for Dnl4 in the maintenance of ribosomal DNA stability.

Replication stress leads to genome instabilities in Arabidopsis DNA polymerase delta mutants.

Impeded DNA replication or a deficiency of its control may critically threaten the genetic information of cells, possibly resulting in genome alterations, such as gross chromosomal translocations, microsatellite instabilities, or increased rates of homologous recombination (HR). We examined an Arabidopsis thaliana line derived from a forward genetic screen, which exhibits an elevated frequency of somatic HR. These HR events originate from replication stress in endoreduplicating cells caused by reduced expression of the gene coding for the catalytic subunit of the DNA polymerase delta (POLdelta1). The analysis of recombination types induced by diverse alleles of poldelta1 and by replication inhibitors allows the conclusion that two not mutually exclusive mechanisms lead to the generation of recombinogenic breaks at replication forks. In plants with weak poldelta1 alleles, we observe genome instabilities predominantly at sites with inverted repeats, suggesting the formation and processing of aberrant secondary DNA structures as a result of the accumulation of unreplicated DNA. Stalled and collapsed replication forks account for the more drastic enhancement of HR in plants with strong poldelta1 mutant alleles. Our data suggest that efficient progression of DNA replication, foremost on the lagging strand, relies on the physiological level of the polymerase delta complex and that even a minor disturbance of the replication process critically threatens genomic integrity of Arabidopsis cells.

Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks.

INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (gammaH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, gammaH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation.

The dual nature of homologous recombination in plants.

Homologous recombination creates covalent linkages between DNA in regions of highly similar or identical sequence. Recent results from several laboratories, many of them based on forward and reverse genetics in Arabidopsis, give insights into the mechanisms of the enzymatic machinery and the involvement of chromatin in somatic and meiotic DNA recombination. Also, signaling pathways and interconnections between repair pathways are being discovered. In addition, recent work shows that biotic and abiotic influences from the environment can dramatically affect plant genomes. The resulting changes in the DNA sequence, exerted at the level of somatic or meiotic tissue, might contribute to evolution.

Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair.

The budding yeast INO80 complex is a conserved ATP-dependent nucleosome remodeler containing actin-related proteins Arp5 and Arp8. Strains lacking INO80, ARP5, or ARP8 have defects in transcription. Here we show that these mutants are hypersensitive to DNA damaging agents and to double-strand breaks (DSBs) induced by the HO endonuclease. The checkpoint response and most transcriptional modulation associated with induction of DNA damage are unaffected by these mutations. Using chromatin immunoprecipitation we show that Ino80, Arp5, and Arp8 are recruited to an HO-induced DSB, where a phosphorylated form of H2A accumulates. Recruitment of Ino80 is compromised in cells lacking the H2A phosphoacceptor S129. Finally, we demonstrate that conversion of the DSB into ssDNA is compromised in arp8 and H2A mutants, which are both deficient for INO80 activity at the site of damage. These results implicate INO80-mediated chromatin remodeling directly at DSBs, where it appears to facilitate processing of the lesion.

The INO80 protein controls homologous recombination in Arabidopsis thaliana.

Homologous recombination (HR) serves a dual role in providing genetic flexibility and in maintaining genome integrity. Little is known about the regulation of HR and other repair pathways in the context of chromatin. We report on a mutant affected in the expression of the Arabidopsis INO80 ortholog of the SWI/SNF ATPase family, which shows a reduction of the HR frequency to 15% of that in wild-type plants. In contrast, sensitivity to genotoxic agents and efficiency of T-DNA integration remain unaffected, suggesting that INO80 is a positive regulator of HR, while not affecting other repair pathways. So far, INO80 function has only been reported in a lower eukaryote. Profiling studies on three ino80 allelic mutants show that INO80 regulates nearly 100 Arabidopsis genes. However, the transcriptional regulation of repair-related genes is unaffected in the mutant. This suggests a dual role for INO80 in transcription and DNA repair by HR.

CENTRIN2 modulates homologous recombination and nucleotide excision repair in Arabidopsis.

A genetic screen of a population of Arabidopsis thaliana lines exhibiting enhanced somatic homologous recombination yielded a mutant affected in expression of a gene encoding a caltractin-like protein (centrin). The hyperrecombinogenic phenotype could be reproduced using RNA interference (RNAi) technology. Both the original mutant and the RNAi plants exhibited a moderate UV-C sensitivity as well as a reduced efficiency of in vitro repair of UV-damaged DNA. Transcription profiling of the mutant showed that expression of components of the nucleotide excision repair (NER) pathway and of factors involved in other DNA repair processes were significantly changed. Our data suggest an indirect involvement of centrin in recombinational DNA repair via the modulation of the NER pathway. These findings thus point to a novel interconnection between an early step of NER and homologous recombination, which may play a critical role in plant DNA repair.