Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

Kramer, Kate; Brock, J A; Bloom, Kerry; Moore, J K; Haber, James E.

doi:10.1128/mcb.14.2.1293

Cited by 197 publications

(181 citation statements)

References 69 publications

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“…Thus, use of the 4Pnt can be considered a hallmark of the canonical NHEJ pathway. In Saccharomyces cerevisiae, imperfect end-joining involves small deletions and fill-ins in a KU-dependent manner (29,30). In accordance with our data, it suggests that extended deletion associated with the use of microhomologies distant from the end is a hallmark of the NHEJ-alt pathway and is thus a process different from that using the 4Pnt, as also suggested by experiments using episomic plasmids (6).…”

Section: Discussionsupporting

confidence: 78%

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Guirouilh‐Barbat

Rass

Plo

et al. 2007

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

152

183

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XRCC4-null mice have a more severe phenotype than KU80-null mice. Here, we address whether this difference in phenotype is connected to nonhomologous end-joining (NHEJ). We used intrachromosomal substrates to monitor NHEJ of two distal doublestrand breaks (DSBs) targeted by I-SceI, in living cells. In xrcc4-defective XR-1 cells, a residual but significant end-joining process exists, which primarily uses microhomologies distal from the DSB. However, NHEJ efficiency was strongly reduced in xrcc4-defective XR-1 cells versus complemented cells, contrasting with KU-deficient xrs6 cells, which showed levels of end-joining similar to those of complemented cells. Nevertheless, sequence analysis of the repair junctions indicated that the accuracy of end-joining was strongly affected in both xrcc4-deficient and KU-deficient cells. More specifically, these data showed that the KU80/XRCC4 pathway is conservative and not intrinsically error-prone but can accommodate non-fully complementary ends at the cost of limited mutagenesis.double-strand break repair ͉ genome rearrangements D NA double-strand breaks (DSBs) are harmful lesions generated by a variety of endogenous or exogenous stresses, potentially leading to genomic rearrangement. Nonhomologous endjoining (NHEJ) is a prominent pathway for DSB repair (1). Canonical NHEJ involves the successive intervention of the KU80-KU70 heterodimer, DNA-PKcs-Artemis, and, finally, ligase IV (Lig4) associated with its cofactors XRCC4 and Cernunnos/Xlf (2, 3). KU-independent NHEJ (KU-alt) has been described in vitro in both acellular extracts and cultured cells (1, 4-6).Alternative, XRCC4-independent DSB repair pathways (XRCC4-alt) have also been described, using episomic plasmid in cultured cells, using pulse field gel electrophoresis, or in in vitro biochemical experiments (5-10). One hypothesis could propose that XRCC4 and KU are implicated in the same canonical NHEJ pathway, whereas the alternate pathway is independent of both KU and XRCC4. However, in transgenic mice, the inactivation of XRCC4 or Lig4 results in a more severe phenotype than the inactivation of KU (11), suggesting that XRCC4 might have an additional essential function; however, studies show that XRCC4 and Lig4 do not have roles outside of NHEJ, whereas in contrast, KU acts in other processes such as transcription, apoptosis, and responses to the cell microenvironment (12)(13)(14).Alternatively, these varying phenotypes in mice may actually result from differences in DSB repair efficiencies, indicating that defects in XRCC4 might be more deleterious for DSB repair than defects in KU. What challenges this hypothesis, however, is that substantial class switch recombination (CSR) has recently been shown to occur in mouse B cells without XRCC4, whereas no CSR was recorded in cells devoid of KU (15-18).The relative contributions of XRCC4 and KU80 versus the XRCC4-alt and KU-alt pathways, respectively, to DSB repair remain unclear in wild-type cells. Contrasting results were obtained in living cells, using an episomic plasm...

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Section: Discussionsupporting

confidence: 78%

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Guirouilh‐Barbat

Rass

Plo

et al. 2007

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

152

183

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“…This complexity and possible redundancy has hindered genetic and biochemical analysis of the end-processing reactions in NHEJ. Many of the end-processing events seem to involve an alignment step in which a short tract of sequence homology at or near the DNA ends is used to align the DNA ends (7,9,10). Whether the short microhomologies are exposed by unwinding or by nucleolytic degradation remains unknown.…”

Section: Discussionmentioning

confidence: 99%

“…However, the termini of the majority of DSBs generated in vivo by agents such as ionizing radiation will be noncomplementary and may also have damaged termini. Analysis of DNA molecules repaired by NHEJ has shown that short complementary sequences, so called microhomologies, are preferentially used for alignment of broken DNA ends (7,9,10). This suggests that, after DNA ends are brought together, the DNA regions next to the ends are compared in some manner for short complementary sequences.…”

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confidence: 99%

Processing and Joining of DNA Ends Coordinated by Interactions among Dnl4/Lif1, Pol4, and FEN-1

Tseng

Tomkinson

2004

Journal of Biological Chemistry

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The repair of DNA double-strand breaks is critical for maintaining genetic stability. In the non-homologous end-joining pathway, DNA ends are brought together by end-bridging factors. However, most in vivo DNA double-strand breaks have terminal structures that cannot be directly ligated. Thus, the DNA ends are aligned using short regions of sequence microhomology followed by processing of the aligned DNA ends by DNA polymerases and nucleases to generate ligatable termini. Genetic studies in Saccharomyces cerevisiae have implicated the DNA polymerase Pol4 and the DNA structurespecific endonuclease FEN-1(Rad27) in the processing of DNA ends to be joined by Dnl4/Lif1. In this study, we demonstrated that FEN-1(Rad27) physically and functionally interacted with both Pol4 and Dnl4/Lif1 and that together these proteins coordinately processed and joined DNA molecules with incompatible 5 ends. Because Pol4 also interacts with Dnl4/Lif1, our results have revealed a series of pair-wise interactions among the factors that complete the repair of DNA doublestrand breaks by non-homologous end-joining and provide a conceptual framework for delineating the endprocessing reactions in higher eukaryotes.The repair of DNA double-strand breaks (DSBs) 1 is critical for the maintenance of genomic integrity and stability. There are two main DSB repair pathways in eukaryotes: homologous recombination and non-homologous end joining (NHEJ) (1). In the error-free homologous recombination pathway, an intact homologous DNA duplex acts as a template for repair, resulting in the accurate restoration of the broken DNA molecule. By contrast, in NHEJ, the two broken ends are simply rejoined to one another in a process that frequently causes loss and/or gain of nucleotides at the break site and occasionally results in the joining of previously unlinked DNA molecules. Notably, defects in the repair of DSBs by either homologous recombination or NHEJ have been linked with cancer predisposition (2, 3).Studies with mammalian cells identified the DNA-dependent protein kinase (DNA-PK), which is composed of the DNA end binding Ku70/Ku80 heterodimer and the DNA-PK catalytic subunit, and the DNA ligase IV/XRCC4 complex as key NHEJ factors (4). In Saccharomyces cerevisiae, Hdf1/Hdf2 and Dnl4/Lif1 are the functional homologs of Ku70/Ku80 and DNA ligase IV/XRCC4, respectively (4). Although yeast lacks a DNA-PK catalytic subunit homolog, genetic studies have revealed that the Rad50/Mre11/Xrs2 complex is a key player in NHEJ (4 -7). Using purified protein complexes, it has been shown that the Rad50/Mre11/Xrs2 complex has robust endbridging activity and specifically stimulates intermolecular DNA joining by Dnl4/Lif1 (8). Furthermore, efficient intermolecular DNA joining mediated by the Rad50/Mre11/Xrs2 and Dnl4/Lif1 complexes was dependent upon Hdf1/Hdf2 at physiological salt concentrations (8). Based on these results, it seems likely that Hdf1/Hdf2 enhances the recruitment of the Rad50/ Mre11/Xrs2 end-bridging complex to DNA ends and that Dnl4/ Lif1 joins the res...

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“…Mechanisms of DNA end joining have been investigated in detail in a variety of eucarytic in vivo (cultured mammalian cells, [13][14][15][16]; Xenopus laevis eggs, 17; yeast, 9,18,19) and in vitro systems (extracts from Xenopus laevis eggs [20][21][22]; nuclear extracts from human cells, [23][24][25][26][27] all of which were shown to be able to join DNA termini containing either blunt ends or short protruding single strands (PSS). Junctional sequence analysis revealed the existence of various different junction types with distinct features which led to the postulation of different pathways of DNA end joining (21,22,25,26,27).…”

Section: Introductionmentioning

confidence: 99%

Nonhomologous DNA end joining of synthetic hairpin substrates inXenopus laevisegg extracts

et al. 1994

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Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'-or 3'-protruding single-strands (PSS) of arbitrary sequence and length.

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Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

Cited by 197 publications

References 69 publications

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Processing and Joining of DNA Ends Coordinated by Interactions among Dnl4/Lif1, Pol4, and FEN-1

Nonhomologous DNA end joining of synthetic hairpin substrates inXenopus laevisegg extracts

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