Nonhomologous End Joining in Yeast

Daley, James M.; Palmbos, Phillip L.; Wu, Dongliang; Wilson, Thomas E.

doi:10.1146/annurev.genet.39.073003.113340

Cited by 371 publications

(306 citation statements)

References 103 publications

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“…Many organisms, including S. cerevisiae, perform nonhomologous end joining (NHEJ) as a way to maintain the integrity of the genome in the presence of double-strand breaks (10). In contrast to homologous recombination, this mechanism for joining DNA does not depend on sequence identity.…”

Section: Analysis Of 10mentioning

confidence: 99%

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

Gibson

Benders

Axelrod

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

411

318

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We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.in vivo DNA assembly ͉ genome synthesis ͉ combinatorial assembly ͉ yeast transformation ͉ Mycoplasma genitalium ͉ synthetic biology Y east has long been considered a genetically tractable organism because of its ability to take up and recombine DNA fragments. More than 30 years ago, Hinnen et al. (1) reported the restoration of leucine biosynthesis in Saccharomyces cerevisiae by transformation of a leu2 -strain to LEU2ϩ using a method involving spheroplasts, CaCl 2 , and PEG. Soon after, Orr-Weaver et al. (2) reported mechanistic studies demonstrating that DNA molecules taken up during yeast transformation can integrate into yeast chromosomes through homologous recombination, and that the ends of the linear-transforming DNA are highly recombinogenic and react directly with homologous chromosomal sequences, whereas circular plasmids carrying yeast sequences integrate by a single crossover and only at low frequency. Subsequently, yeast transformation has become an indispensable tool in yeast genetics.Yeast recombination has since been applied to the construction of plasmids and yeast artificial chromosomes (YACs). In 1987, Ma et al. (3) constructed plasmids from two cotransformed DNA fragments containing homologous regions. In another process, called linker-mediated assembly, any DNA sequence can be joined to a vector DNA using short synthetic linkers that bridge the ends (4, 5). Similarly, four or five overlapping DNA pieces can be assembled and joined to vector DNA (4, 6, 7). This work demonstrated that yeast cells can take up multiple pieces of DNA, and that homologous yeast recombination is sufficiently efficient to correctly assemble the pieces into a single recombinant molecule.The limitations of assembly methods in yeast remain unknown. We recently assembled an entire synthetic M. genitalium genome using a combination of in vitro enzymatic recombination in early stages and in vivo yeast recombination in the final stage to produce the complete genome (8). In the first stage, overlapping Ϸ6-kb DNA cassettes were joined four at a time to form 25 Ϸ24-kb A-series assemblies. Three A-series assemblies were then joined to make 1/8 genome Ϸ72-kb B-series assemblies, and then two Bseries assemblies were assembled to make each of the Ϸ145-kb quarter-genome C-series assemblies. We accomplished the final assembly in yeast using three quarter-genome fragments and a fourth quarter fragment that had been cleaved by a restriction enzyme to provide a site for insertion of the vector DNA. Thus, some individual yeast cells have the capacity to simultaneously tak...

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Section: Analysis Of 10mentioning

confidence: 99%

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

Gibson

Benders

Axelrod

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

411

318

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“…4 Ligation of the broken DNA ends is accomplished by the Lig4/Lif1 complex in S. cerevisiae and Lig4/XRCC4 and XLF/Cernunnos factors in mammals. [5][6][7] Due to nucleolytic processing of DNA ends to make them compatible for subsequent ligation, NHEJ can result in short deletions, and thus this process is error-prone. 4 Homologous recombination (HR) functions primarily in the S/G 2 phase of the cell cycle, and it relies on sequence homology from an undamaged sister chromatid or a homologous DNA sequence to use as a template for copying the missing information.…”

Section: Introductionmentioning

confidence: 99%

Opening the DNA repair toolbox: Localization of DNA double strand breaks to the nuclear periphery

Oza¹,

Peterson²

2010

Cell Cycle

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E fficient repair of DNA double strand breaks is essential for cells to avoid increased mutation rates, genomic instability, and even cell death. Consequently, cells have evolved multiple mechanisms for rapidly repairing these DNA lesions, including error-free homologous recombination as well as error-prone pathways such as nonhomologous end joining. What happens to DSBs that are repaired inefficiently or not at all? Recently, several studies in budding yeast have shown that these more recalcitrant DSBs are localized to the nuclear periphery through interactions between the nuclear envelope protein, Mps3, and proteins associated with DSB chromatin. Why these DSBs are tethered to the nuclear periphery is still not clear, though the current view is that alternative repair pathways may be activated at the periphery in a final attempt to repair the lesion. In this Extra View, we discuss these recent reports, and we show that the Est1 component of the telomerase machinery plays an essential role in anchoring DSB chromatin to the nuclear envelope protein, Mps3.

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“…Nonhomologous end joining (NHEJ) is a mode of DNA double-strand break (DSB) repair in which the two DNA ends are directly rejoined [1][2][3]. The critical step in NHEJ is strand ligation.…”

Section: Introductionmentioning

confidence: 99%

Modes of interaction among yeast Nej1, Lif1 and Dnl4 proteins and comparison to human XLF, XRCC4 and Lig4

Deshpande

Wilson

2007

DNA Repair

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The nonhomologous end joining (NHEJ) pathway of double-strand break repair depends on DNA ligase IV and its interacting partner protein XRCC4 (Lif1 in yeast). A third yeast protein, Nej1, interacts with Lif1 and supports NHEJ, similar to the distantly related mammalian Nej1 orthologue XLF (also known as Cernunnos). XRCC4/Lif1 and XLF/Nej1 are themselves related and likely fold into similar coiled-coil structures, which suggests many possible modes of interaction between these proteins. Using yeast two-hybrid and co-precipitation methods we examined these interactions and the protein domains required to support them. Results suggest that stable coiled-coil homodimers are a predominant form of XLF/Nej1, just as for XRCC4/Lif1, but that similar heterodimers are not. XLF-XRCC4 and Nej1-Lif1 interactions were instead mediated independently of the coiled coil, and by different regions of XLF and Nej1. Specifically, the globular head of XRCC4/Lif1 interacted with N-and C-terminal domains of XLF and Nej1, respectively. Direct interactions between XLF/Nej1 and DNA ligase IV were also observed, but again appeared qualitatively different than the stable coiled coil-mediated interaction between XRCC4/Lif1 and DNA ligase IV. The implications of these findings for DNA ligase IV function are considered in light of the evolutionary pattern in the XLF/ XRCC4 and XLF/Nej1 family.

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Nonhomologous End Joining in Yeast

Cited by 371 publications

References 103 publications

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

Opening the DNA repair toolbox: Localization of DNA double strand breaks to the nuclear periphery

Modes of interaction among yeast Nej1, Lif1 and Dnl4 proteins and comparison to human XLF, XRCC4 and Lig4

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