Genomic DNA is transiently contaminated with ribonucleotide residues during the process of DNA replication through misincorporation by the replicative DNA polymerases α, δ and ε, and by the normal ...replication process on the lagging strand, which uses RNA primers. These ribonucleotides are efficiently removed during replication by RNase H enzymes and the lagging strand synthesis machinery. However, when ribonucleotides remain in DNA they can distort the DNA helix, affect machineries for DNA replication, transcription and repair, and can stimulate genomic instabilities which are manifest as increased mutation, recombination and chromosome alterations. The genomic instabilities associated with embedded ribonucleotides are considered here, along with a discussion of the origin of the lesions that stimulate particular classes of instabilities.
Homologous recombination (HR) is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome ...maintenance. As such, HR is indispensable for genome integrity, but it must be regulated to avoid deleterious events. Mutations in the tumour-suppressor protein BRCA2, which has a mediator function in HR, lead to cancer formation. DNA helicases, such as Bloom's syndrome protein (BLM), regulate HR at several levels, in attenuating unwanted HR events and in determining the outcome of HR. Defects in BLM are also associated with the cancer phenotype. The past several years have witnessed dramatic advances in our understanding of the mechanism and regulation of HR.
The Rad51 recombinase is an essential factor for homologous recombination and the repair of DNA double strand breaks, binding transiently to both single stranded and double stranded DNA during the ...recombination reaction. The use of a homologous recombination mechanism to repair DNA damage is controlled at several levels, including the binding of Rad51 to single stranded DNA to form the Rad51 nucleofilament, which is controlled through the action of DNA helicases that can counteract nucleofilament formation. Overexpression of Rad51 in different organisms and cell types has a wide assortment of consequences, ranging from increased homologous recombination and increased resistance to DNA damaging agents to disruption of the cell cycle and apoptotic cell death. Rad51 expression is increased in p53-negative cells, and since p53 is often mutated in tumor cells, there is a tendency for Rad51 to be overexpressed in tumor cells, leading to increased resistance to DNA damage and drugs used in chemotherapies. As cells with increased Rad51 levels are more resistant to DNA damage, there is a selection for tumor cells to have higher Rad51 levels. While increased Rad51 can provide drug resistance, it also leads to increased genomic instability and may contribute to carcinogenesis.
Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of ...replication forks, for telomere maintenance, and chromosome segregation in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facilitate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.
Srs2 helicase is known to dismantle nucleofilaments of Rad51 recombinase to prevent spurious recombination events and unwind trinucleotide sequences that are prone to hairpin formation. Here we ...document a new, unexpected genome maintenance role of Srs2 in the suppression of mutations arising from mis-insertion of ribonucleoside monophosphates during DNA replication. In cells lacking RNase H2, Srs2 unwinds DNA from the 5' side of a nick generated by DNA topoisomerase I at a ribonucleoside monophosphate residue. In addition, Srs2 interacts with and enhances the activity of the nuclease Exo1, to generate a DNA gap in preparation for repair. Srs2-Exo1 thus functions in a new pathway of nick processing-gap filling that mediates tolerance of ribonucleoside monophosphates in the genome. Our results have implications for understanding the basis of Aicardi-Goutières syndrome, which stems from inactivation of the human RNase H2 complex.
Ribonucleotides can be incorporated into DNA during replication by the replicative DNA polymerases. These aberrant DNA subunits are efficiently recognized and removed by Ribonucleotide Excision ...Repair, which is initiated by the heterotrimeric enzyme RNase H2. While RNase H2 is essential in higher eukaryotes, the yeast Saccharomyces cerevisiae can survive without RNase H2 enzyme, although the genome undergoes mutation, recombination and other genome instability events at an increased rate. Although RNase H2 can be considered as a protector of the genome from the deleterious events that can ensue from recognition and removal of embedded ribonucleotides, under conditions of high ribonucleotide incorporation and retention in the genome in a RNase H2-negative strain, sudden introduction of active RNase H2 causes massive DNA breaks and genome instability in a condition which we term 'ribodysgenesis'. The DNA breaks and genome instability arise solely from RNase H2 cleavage directed to the ribonucleotide-containing genome. Survivors of ribodysgenesis have massive loss of heterozygosity events stemming from recombinogenic lesions on the ribonucleotide-containing DNA, with increases of over 1000X from wild-type. DNA breaks are produced over one to two divisions and subsequently cells adapt to RNase H2 and ribonucleotides in the genome and grow with normal levels of genome instability.
In the current view of DNA replication, polymerase (Pol) is the major leading-strand DNA polymerase (DNAP) and Pol the major lagging-strand DNAP, with Pol-primase initiating the ends of new DNA ...fragments (Fig. 1). It is critical that DNAPs select the base that pairs properly with the template but also that the selected base be attached to the correct sugar. It has only recently been appreciated that incorporation of ribonucleoside monophosphates (rNMPs) in place of the corresponding deoxyribonucleoside monophosphates is widespread and associated with unwanted genetic consequences. How, when, where and why does rNMP incorporation occur during replication? Four papers independently address the where by mapping rNMP locations genome wide in budding and fission yeasts: Clausen et al. and Daigaku et al. in this issue of Nature Structural & Molecular Biology, Reijns et al. in Nature and Koh et al. in Nature Methods. Each study is a technical tour de force, and each provides new and important insight into the complex enzymology of eukaryotic DNA replication.
Ribonucleotides are misincorporated into replicating DNA due to the similarity of deoxyribonucleotides and ribonucleotides, the high concentration of ribonucleotides in the nucleus and the imperfect ...accuracy of replicative DNA polymerases in choosing the base with the correct sugar. Embedded ribonucleotides change certain properties of the DNA and can interfere with normal DNA transactions. Therefore, misincorporated ribonucleotides are targeted by the cell for removal. Failure to remove ribonucleotides from DNA results in an increase in genome instability, a phenomenon that has been characterized in various systems using multiple assays. Recently, however, another side to ribonucleotide misincorporation has emerged, where there is evidence for a functional role of misinserted ribonucleotides in DNA, leading to beneficial consequences for the cell. This review examines examples of both positive and negative effects of genomic ribonucleotide misincorporation in various organisms, aiming to highlight the diversity and the utility of this common replication variation.
Copy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae ...monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC formation is replicative, separable from repeat loss, and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Last, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.
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•rDNA circles form through a replicative mechanism independent of array instability•Production of rDNA circles anti-correlates with total rDNA copy number•Steady-state level of rDNA circles is regulated by diet and rRNA production•rDNA circles re-insert into the chromosomal rDNA array
Copy number variation is associated with both disease and chromosomal evolution. Mansisidor et al. show that healthy yeast cells continuously monitor the copy number of ribosomal DNA repeats and that one strategy for balancing copy number loss is through the amplification of repeats as DNA circles.
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