Abstract
Although fusions between the centromeres of different human chromosomes have been observed cytologically in cancer cells, since the centromeres are long arrays of satellite sequences, the ...details of these fusions have been difficult to investigate. We developed methods of detecting recombination within the centromeres of the yeast Saccharomyces cerevisiae (intercentromere recombination). These events occur at similar rates (about 10−8/cell division) between two active or two inactive centromeres. We mapped the breakpoints of most of the recombination events to a region of 43 base pairs of uninterrupted homology between the two centromeres. By whole-genome DNA sequencing, we showed that most (>90%) of the events occur by non-reciprocal recombination (gene conversion/break-induced replication). We also found that intercentromere recombination can involve non-homologous chromosome, generating whole-arm translocations. In addition, intercentromere recombination is associated with very frequent chromosome missegregation. These observations support the conclusion that intercentromere recombination generally has negative genetic consequences.
Graphical Abstract
Graphical Abstract
Oxidative stress has been implicated in a variety of human diseases. One plausible mechanism is that reactive active species can induce DNA damages and jeopardize genome integrity. To explore how ...oxidative stress results in global genomic instability in cells, our current study examined the genomic alterations caused by H
2
O
2
exposure at the whole genome level in yeast. Using SNP microarrays and genome sequencing, we mapped H
2
O
2
-induced genomic alterations in the yeast genome ranging from point mutations and mitotic recombination to chromosomal aneuploidy. Our results suggested most H
2
O
2
-induced mitotic recombination events were the result of DNA double-stand breaks generated by hydroxyl radicals. Moreover, the mutagenic effect of H
2
O
2
was shown to be largely dependent on DNA polymerase ζ. Lastly, we showed that H
2
O
2
exposure allows rapid phenotypic evolution in yeast strains. Our findings indicate DNA lesions resulting from H
2
O
2
may be general factors that drive genome instability and phenotypic evolution in organisms.
Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the ...rates of all of these genetic changes in a diploid strain of Saccharomyces cerevisiae by whole-genome sequencing of many independent isolates (n = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n = 1,337) and mitotic recombination events (n = 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.
Although homologous recombination between transposable elements can drive genomic evolution in yeast by facilitating chromosomal rearrangements, the details of the underlying mechanisms are not fully ...clarified. In the genome of the yeast Saccharomyces cerevisiae, the most common class of transposon is the retrotransposon Ty1. Here, we explored how Cas9-induced double-strand breaks (DSBs) directed to Ty1 elements produce genomic alterations in this yeast species. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements such as deletions, duplications and translocations. In addition, we found elevated rates of mitotic recombination, resulting in loss of heterozygosity. Using Southern analysis coupled with short- and long-read DNA sequencing, we revealed important features of recombination induced in retrotransposons. Almost all of the chromosomal rearrangements reflect the repair of DSBs at Ty1 elements by non-allelic homologous recombination; clustered Ty elements were hotspots for chromosome rearrangements. In contrast, a large proportion (about three-fourths) of the allelic mitotic recombination events have breakpoints in unique sequences. Our analysis suggests that some of the latter events reflect extensive processing of the broken ends produced in the Ty element that extend into unique sequences resulting in break-induced replication. Finally, we found that haploid and diploid strain have different preferences for the pathways used to repair double-stranded DNA breaks. Our findings demonstrate the importance of DNA lesions in retrotransposons in driving genome evolution.
DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates ...of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G₂ phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.
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•Citridones H-L were isolated from a marine-derived fungus Penicillium sp.•Citridone H contained a rare 6-6/6 ring system of phenyl-pyranopyridone.•Citridone J contained a unique ...phenyl-spiro cyclopentene-furopyridone.•Citridone I displayed anti-inflammatory activity.
Five new pyridone alkaloids Citridones H-L (1–5) along with one new natural product ent-Citridone A (6) were isolated from the marine-derived fungus Penicillium sp. XZD3-3, which came from the gut of a marine shrimp. The structures and absolute configurations of these compounds were elucidated on the basis of NMR, HRESIMS, UV, modified Mosher′s method, single-crystal X-ray diffraction, and ECD calculation. Compounds 1 and 3 contained unprecedented polycyclic systems of phenyl-pyranopyridone and phenyl-spirocyclopentene-furopyridone, respectively. Compound 2 showed moderate inhibition activity against nitric oxide production with IC50 value of 52.5 μM. All tested compounds were inactive in neither antibacterial nor cytotoxicity assays.
Most cells of solid tumors have very high levels of genome instability of several different types, including deletions, duplications, translocations, and aneuploidy. Much of this instability appears ...induced by DNA replication stress. As a model for understanding this type of instability, we have examined genome instability in yeast strains that have low levels of two of the replicative DNA polymerases: DNA polymerase α and DNA polymerase δ (Polα and Polδ). We show that low levels of either of these DNA polymerases results in greatly elevated levels of mitotic recombination, chromosome rearrangements, and deletions/duplications. The spectrum of events in the two types of strains, however, differs in a variety of ways. For example, a reduced level of Polδ elevates single-base alterations and small deletions considerably more than a reduced level of Polα. In this review, we will summarize the methods used to monitor genome instability in yeast, and how this analysis contributes to understanding the linkage between genome instability and DNA replication stress.
Abstract
Oxidative DNA damage is a threat to genome stability. Using a genetic system in yeast that allows detection of mitotic recombination, we found that the frequency of crossovers is greatly ...elevated when cells are treated with hydrogen peroxide (H2O2). Using a combination of microarray analysis and genomic sequencing, we mapped the breakpoints of mitotic recombination events and other chromosome rearrangements at a resolution of about 1 kb. Gene conversions and crossovers were the two most common types of events, but we also observed deletions, duplications, and chromosome aneuploidy. In addition, H2O2-treated cells had elevated rates of point mutations (particularly A to T/T to A and C to G/G to C transversions) and small insertions/deletions (in/dels). In cells that underwent multiple rounds of H2O2 treatments, we identified a genetic alteration that resulted in improved H2O2 tolerance by amplification of the CTT1 gene that encodes cytosolic catalase T. Lastly, we showed that cells grown in the absence of oxygen have reduced levels of recombination. This study provided multiple novel insights into how oxidative stress affects genomic instability and phenotypic evolution in aerobic cells.
Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of ...mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length.
A Gram-negative, rod-shaped and aerobic bacterial strain B3.7
T
, was isolated from the sediment of Zhairuo Island, Zhoushan city, Zhejiang Province, PR China. Maximum growth of strain B3.7
T
was ...observed at 30 °C when cultured in a medium containing 0.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain B3.7
T
belonged to the genus
Shinella
; it showed the highest sequence similarity of 98.47 % to
Shinella kummerowiae
CCBAU 25048
T
. The average nucleotide identity and digital DNA–DNA hybridization values between strain B3.7
T
and its reference strains were 82.9–84.2 % and 26.1–27.3 %, respectively. Chemotaxonomic analysis indicated that the sole respiratory quinone was Q-10 and the predominant cellular fatty acids were C
19 : 0
cyclo
ω
8
c
, C
16 : 0
, C
18 : 1
ω
7
c
11-methyl and summed feature 8 (C
18 : 1
ω
7
c
and/or C
18 : 1
ω
6
c
). The polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids and two unidentified aminolipids. Collectively, strain B3.7
T
can be considered to represent a novel species, for which the name
Shinella sedimenti
sp. nov. is proposed. The type strain is B3.7
T
(=MCCC 1K07163
T
=LMG 32559
T
).
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