Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells

Heidenreich, Erich; Novotny, René; Kneidinger, Bernd; Holzmann, Veronika; Wintersberger, Ulrike

doi:10.1093/emboj/cdg203

Cited by 117 publications

(101 citation statements)

References 42 publications

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“…n, proportion of indels among revertants sequenced. mononucleotide runs were previously reported among lys2DBgl revertants isolated in one WT strain background (Heidenreich et al 2003), but this particular class was not observed in at least two other backgrounds (Marsischky et al 1996;Greene and Jinks-Robertson 1997). In the lys2DA746,NR assay, 2-bp deletions and 4-bp tandem duplications each comprised 10% of the reversion spectrum, and each class was significantly reduced in the dnl4D background.…”

Section: Discussionmentioning

confidence: 67%

Frameshift Mutagenesis: The Roles of Primer–Template Misalignment and the Nonhomologous End-Joining Pathway inSaccharomyces cerevisiae

Lehner¹,

Mudrak

Minesinger

et al. 2012

Genetics

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Small insertions or deletions that alter the reading frame of a gene typically occur in simple repeats such as mononucleotide runs and are thought to reflect spontaneous primer-template misalignment during DNA replication. The resulting extrahelical repeat is efficiently recognized by the mismatch repair machinery, which specifically replaces the newly replicated strand to restore the original sequence. Frameshift mutagenesis is most easily studied using reversion assays, and previous studies in Saccharomyces cerevisiae suggested that the length threshold for polymerase slippage in mononucleotide runs is 4N. Because the probability of slippage is strongly correlated with run length, however, it was not clear whether shorter runs were unable to support slippage or whether the resulting frameshifts were obscured by the presence of longer runs. To address this issue, we removed all mononucleotide runs .3N from the yeast lys2DBgl and lys2DA746 frameshift reversion assays, which detect net 1-bp deletions and insertions, respectively. Analyses demonstrate that 2N and 3N runs can support primer-template misalignment, but there is striking run-specific variation in the frequency of slippage, in the accumulation of +1 vs. 21 frameshifts and in the apparent efficiency of mismatch repair. We suggest that some of this variation reflects the role of flanking sequence in initiating primer-template misalignment and that some reflects replication-independent frameshifts generated by the nonhomologous end-joining pathway. Finally, we demonstrate that nonhomologous end joining is uniquely required for the de novo creation of tandem duplications from noniterated sequence.T HE accumulation of mutations within genomic DNA is precisely regulated; mutations must be kept at a very low level to maintain genome integrity and yet must be frequent enough to support evolutionary change. Most spontaneous mutations are base substitutions or small insertions/deletions (indels) that reflect errors made either when replicating an undamaged DNA template or when synthesizing over a DNA lesion. Indels that are not a multiple of 3 bp are referred to as frameshift mutations because they change the reading frame of a translating ribosome, thereby altering all downstream amino acids and usually resulting in premature termination of translation. Given the very deleterious nature of frameshift mutations, it is critical that the corresponding mutational intermediates be efficiently recognized and removed.Repetitive sequences such as mononucleotide or dinucleotide repeats are strong hotspots for frameshifts, and most intermediates arise through spontaneous, replication-associated strand slippage (Streisinger et al. 1966). As illustrated for a mononucleotide run in Figure 1A, misalignment between the primer and template strands generates an extrahelical repeat on one of the two strands. If not repaired, an extrahelical nucleotide on the primer strand will become a +1 frameshift mutation, while the persistence of an extrahelical nucleotide on the te...

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Section: Discussionmentioning

confidence: 67%

Frameshift Mutagenesis: The Roles of Primer–Template Misalignment and the Nonhomologous End-Joining Pathway inSaccharomyces cerevisiae

Lehner¹,

Mudrak

Minesinger

et al. 2012

Genetics

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“…In addition, mitochondria-containing vacuolar autophagic bodies accumulate in budding yeast stationary phase (Takeshigeet al 1992), providing a mechanism for increased release of mtDNA molecules in the cytoplasm (Campbell and Thorsness 1998). Alternatively, increased mtDNA capture frequency may be due to enhanced NHEJ efficiency in glucose-starved cells as suggested by studies of budding yeast (Karathanasis and Wilson 2002;Heidenreich et al 2003). Finally, it should be emphasized that yeast cells grown to stationary phase resemble most of the cells from multicellular organisms since (1) most energy comes from mitochondrial respiration and (2) cells have exited from the cell cycle; i.e., they have entered the G 0 phase.…”

Section: Discussionmentioning

confidence: 96%

Capture of Extranuclear DNA at Fission Yeast Double-Strand Breaks

Decottignies

2005

Genetics

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Proper repair of DNA double-strand breaks (DSBs) is necessary for the maintenance of genomic integrity. Here, a new simple assay was used to study extrachromosomal DSB repair in Schizosaccharomyces pombe. Strikingly, DSB repair was associated with the capture of fission yeast mitochondrial DNA (mtDNA) at high frequency. Capture of mtDNA fragments required the Lig4p/Pku70p nonhomologous end-joining (NHEJ) machinery and its frequency was highly increased in fission yeast cells grown to stationary phase. The fission yeast Mre11 complex Rad32p/Rad50p/Nbs1p was also required for efficient capture of mtDNA at DSBs, supporting a role for the complex in promoting intermolecular ligation. Competition assays further revealed that microsatellite DNA from higher eukaryotes was preferentially captured at yeast DSBs. Finally, cotransformation experiments indicated that, in NHEJ-deficient cells, capture of extranuclear DNA at DSBs was observed if homologies-as short as 8 bp-were present between DNA substrate and DSB ends. Hence, whether driven by NHEJ, microhomology-mediated end-joining, or homologous recombination, DNA capture associated with DSB repair is a mutagenic process threatening genomic stability.

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“…Recombination could influence genetic variation directly; for example, DSBs both require DNA synthesis for repair (hence can potentially introduce copying errors) and may also expose DNA to cellular mutagens. Evidence for a mutagenic effect of recombination comes from both direct experiment [31][32][33][34] and analysis of rates of molecular evolution [6,9,11,35].…”

Section: Recombination Hotspots Have a Direct Effect On The Frequencymentioning

confidence: 99%

The influence of recombination on human genetic diversity

Spencer¹,

Deloukas²,

Hunt³

et al. 2005

PLoS Genet

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In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.

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Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells

Cited by 117 publications

References 42 publications

Frameshift Mutagenesis: The Roles of Primer–Template Misalignment and the Nonhomologous End-Joining Pathway inSaccharomyces cerevisiae

Frameshift Mutagenesis: The Roles of Primer–Template Misalignment and the Nonhomologous End-Joining Pathway inSaccharomyces cerevisiae

Capture of Extranuclear DNA at Fission Yeast Double-Strand Breaks

The influence of recombination on human genetic diversity

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