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Quantitative Insights into Age-Associated DNA-Repair Inefficiency in Single Cells

aging system biology dna repair double strand break replicative lifespan single cell yeast single-strand annealing microscopy microfluidics

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#1 Engadin

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Posted 30 August 2019 - 12:59 PM


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S O U R C E :   Cell

 

 

 

HIGHLIGHTS

 

  • Efficiency of DNA repair by single-strand annealing is lower in old yeast cells
  • This decline is not due to increased use of non-homologous end joining
  • Cell-cycle regulation is involved in the age-associated DNA repair inefficiency
  • Heteroduplex rejection remains high in old cells

 

 

 

SUMMARY

 

Although double-strand break (DSB) repair is essential for a cell’s survival, little is known about how DSB repair mechanisms are affected by age. Here we characterize the impact of cellular aging on the efficiency of single-strand annealing (SSA), a DSB repair mechanism. We measure SSA repair efficiency in young and old yeast cells and report a 23.4% decline in repair efficiency. This decline is not due to increased use of non-homologous end joining. Instead, we identify increased G1 phase duration in old cells as a factor responsible for the decreased SSA repair efficiency. Expression of 3xCLN2 leads to higher SSA repair efficiency in old cells compared with expression of 1xCLN2, confirming the involvement of cell-cycle regulation in age-associated repair inefficiency. Examining how SSA repair efficiency is affected by sequence heterology, we find that heteroduplex rejection remains high in old cells. Our work provides insights into the links between single-cell aging and DSB repair efficiency.

 

 

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DNA damage has long been hypothesized to be both a driver and a consequence of aging (Burhans and Weinberger, 2007, Freitas and de Magalhães, 2011). Old tissues and cells accumulate DNA damage and mutations (McMurray and Gottschling, 2003, Adams et al., 2015, Lu et al., 2004). Such mutations can negatively affect tissue homeostasis and organismal function and may even increase the risk for cancer (McMurray and Gottschling, 2004, Aparicio et al., 2014, White et al., 2015). The DNA double-strand break (DSB) represents a major category of DNA damage. Inability to repair a DSB can lead to cell death or genomic instability (Morgan et al., 1998). Even when a DSB is repaired, there are mutagenic repair mechanisms that can both increase genetic variability and affect cellular fitness (Symington et al., 2014). Because of the potential for DSB repair to affect so many cellular functions that intersect with the aging process, the question of how cellular aging affects DSB repair efficiency in single cells is an important area of research with several open questions.
 
A useful model system to understand how cellular aging affects DSB repair efficiency is that of replicative aging of the budding yeast Saccharomyces cerevisiae (Steinkraus et al., 2008). Replicative lifespan is the number of times a yeast mother cell produces daughters (Steinkraus et al., 2008). This number varies across genetic backgrounds and growth conditions and is connected to several metabolic pathways (McCormick et al., 2015, Kaeberlein et al., 2005). The connection between DSB repair and replicative lifespan is important for several reasons. Mutations in DSB repair genes result in shortened replicative lifespan, so proper repair of DSBs is necessary for a normal replicative lifespan (Delaney et al., 2013). In the other direction, continued cell division after mutagenic DSB repair results in propagation of these mutations to future generations with potentially negative consequences on cell fitness.
 
Multiple mechanisms exist to repair DSBs, and their efficiency and ability to function properly could change with replicative age. Among these mechanisms, the non-homologous end joining (NHEJ) pathway involves ligation of the free ends flanking the DSB (Ceccaldi et al., 2016). The other main class of DSB repair mechanisms is the set of homology-directed repair (HDR) mechanisms, which make use of sequence homology between the break site and a repair template. Regulation of HDR can have significant consequences for the genome. This is due to the ability of different HDR mechanisms to change allele copy numbers and lead to recombination between chromosomes (Symington et al., 2014). The relative use and efficiency of different DSB repair mechanisms depend on factors including cell-cycle stage, ploidy, and cell type (Ceccaldi et al., 2016, Kadyk and Hartwell, 1992, Trovesi et al., 2011, Karanam et al., 2012). Previous studies have detected changes in repair pathway use between chronologically old and young tissues, which could indicate age-related changes in repair efficiency (Preston et al., 2006, Delabaere et al., 2017, Sukup-Jackson et al., 2014). Other studies have also reported declines in the efficiency and fidelity of NHEJ and HDR in senescent mammalian cells (Seluanov et al., 2004, Mao et al., 2012). However, how the repair efficiency of specific DSB repair pathways longitudinally changes in mitotically aging single cells remains unexplored.
 
Here we assess whether the efficiency of DSB repair via the single-strand annealing (SSA) pathway changes with the age of the host cell. The SSA pathway repairs DSBs occurring between direct repeats of an identical sequence, resulting in deletion of the intermediate sequence (Figure 1A) (Ceccaldi et al., 2016). Repetitive sequences play important roles in cellular function, with the rDNA locus being one prominent example (Sinclair and Guarente, 1997, Paredes and Maggert, 2009). An age-related change in the efficiency of SSA, which would lead to differences in the copy numbers of the repeated sequence, would be expected to have important consequences for the cell. Because SSA and other HDR pathways share regulatory aspects such as end resection and Rad52 recruitment to the repair sites, understanding how SSA efficiency changes with age can provide key insight into whether the efficiency of other HDR mechanisms would also change with age. Using a single-cell longitudinal approach (Song et al., 2018) in a haploid genetic background, we measure the efficiency of SSA repair in young and older cells. We further explore age-related changes in SSA repair efficiency as they relate to age-related changes in cell cycle and NHEJ pathway activity and in terms of the amount of heterology between the SSA repeats.
 

 

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(A) Diagram showing the steps of single-strand annealing based repair. The direct repeats are highlighted in orange. The upper row shows the situation immediately after the double-strand break.
(B) Schematic showing the setup of the experiment. The yeast replicator device allows replicative aging of yeast to be observed using a microscope. Fresh media is supplied, and waste is removed over the course of the movie. A microscope image of a trapped yeast cell is shown above the chip.
© Genetic constructs used to measure SSA. rtTA is expressed from the constitutive PMYO2 promoter. In the presence of doxycycline, rtTA activates expression of I-SceI from a non-leaky PTETO4 promoter. The I-SceI cut site consists of a pair of inverted 18 bp I-SceI sites (black arrows) placed within the SSA reporter (rc, reverse complemented). The non-fluorescent 5′ YFP repeat contains only the 5′ 192 bp of YFP. The 3′ YFP repeat is not expressed because it consists of the entire YFP ORF except for the start codon. Between the TEF1 terminator and 3′ YFP repeat is a 2.1 kb stretch of DNA (dotted line), with inverted I-SceI cut sites at a distance of 0.5 kb from the TEF1 terminator. Between the I-SceI cut site and the 3′ non-functional YFP is an ADH1promoter driving mCherry (abbreviated as “RFP”). Degron-tagged RFP expression below a threshold was used as a reporter for DNA cutting. After SSA repair occurs between the repeats, the resulting product is a full-length YFP with a start codon.
 
 
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Also tagged with one or more of these keywords: aging, system biology, dna repair, double strand break, replicative lifespan, single cell, yeast, single-strand annealing, microscopy, microfluidics

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