Repair of single nucleotide DNA mismatches transfected into mammalian cells can occur by short-patch excision

Heywood, Louise A.; Burke, Julian F.

doi:10.1016/0921-8777(90)90033-2

Cited by 9 publications

(4 citation statements)

References 21 publications

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“…Our conclusions are based on experiments in vitro and are in agreement with the expected preference for initiation of correction of deaminated 5meC. It remains unclear why repair of heteroduplex DNA transfected into mammalian cells exhibits a strong bias in correction of G-T to G:C that is apparently independent of the sequence context of the G-T mispair (Brown & Jiricny, 1987;Heywood & Burke, 1990).…”

Section: Discussionsupporting

confidence: 84%

Incision at DNA G.cntdot.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.cntdot.T binding reaction

Griffin¹,

Karran²

1993

Biochemistry

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We have investigated the specificities of G.T mismatch binding proteins and of G.T mismatch cleavage in extracts of mammalian cells. G.T mismatch-specific protein:DNA complex formation by cell extracts was independent of the local sequence context of the mismatch. Cell extracts performed similar levels of protein binding to DNA substrates in which a single G.T mispair was preceded by T, G, A, C, or 5-meC. In contrast, incision by extracts of the T-containing strand of a G.T mismatch exhibited a strong sequence specificity and efficient strand cleavage was only observed when the mismatched G was in a CpG sequence. Thus, oligonucleotides containing either CpgGpT or 5meCpGGpT were efficiently incised, but not those containing GpGCpT, ApGTpT, or TpGApT sequences. Cell lines made resistant to the alkylating agent N-methyl-N-nitrosourea have previously been found to be defective in a G.T mismatch binding reaction. The defect in binding by extracts prepared from these cells extended to G.T mismatches in several sequence contexts. The variant extracts nevertheless incised G.T mismatches normally suggesting that this particular binding activity is not required for incision. The data indicate that incision by this activity is targeted to the CpG sequences in which G.T mismatches are formed by the mutagenic deamination of DNA 5-methylcytosine. In this regard the repair pathway resembles the very short patch (vsp) repair pathway in Escherichia coli.

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Section: Discussionsupporting

confidence: 84%

Incision at DNA G.cntdot.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.cntdot.T binding reaction

Griffin¹,

Karran²

1993

Biochemistry

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“…Previous work in our laboratory with related plasmid substrates has indicated that the homologous interaction probably precedes the nonhomologous interaction (37). Thus (18) suggested that strand break-dependent strand bias may require the binding of a single-strand endonuclease to mismatches before it cleaves DNA, or that a protein bound to the mismatch interacts with the endonuclease. We observed efficient repair of each of the mismatches created in the present study, including 14-base loops and two single-base mismatches separated by two base pairs.…”

Section: Discussionmentioning

confidence: 99%

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

Deng

Nickoloff

1994

Mol. Cell. Biol.

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Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we DSBs stimulate homologous recombination in yeast cells (22,23,36,38,42; reviewed in reference 48) and mammalian cells (9-11, 21, 24-28, 37, 46, 50, 52; reviewed in reference 8). DNA-damaging agents, such as UV light, X rays, and chemicals, also stimulate recombination (4-6, 13, 16, 31, 33, 43, 51, 53). On the basis of these observations, several recombination models were proposed to explain the recombinogenic effects of DSBs. The DSB repair model (49) nonhomologous regions and the predominance of nonconservative crossover products (1,11,25,27,28,45, 52). Although there have been reports of pathways distinct from SSA in mammalian cells (3, 55), most extrachromosomal recombination can be explained by this model.In this report, we describe our studies of extrachromosomal recombination between heteroallelic neomycin (neo) genes on shuttle vectors in Chinese hamster ovary (CHO) cells. Our system permits an examination of the formation and repair of mismatches in hDNA recombination intermediates. At least 80% of mismatches present in hDNA were repaired prior to DNA replication, and in most crosses tested, a strong repair bias was observed. MATERIALS AND METHODSPlasmid DNA constructions and preparation. Plasmids were constructed and prepared by standard procedures (44) and are shown in Fig. 1. Plasmid SVBss was described previously (37). Plasmid SVBss-XE, containing two mutations in neo, was constructed by inserting a 10-bp XhoI linker into the EagI site of SVBss.The 2.1-kbp HindIII-BamHI fragment of pSV2neo (47) was inserted into the HindIII and BamHI sites of a pUC19 derivative missing the EcoRI site (pUC19-RI), creating pneoAn. Silent mutations, converting the neo EagI site into a PstI site, were introduced into pSV2neo, SVBss, and pneoAn by site-directed mutagenesis (12) to create plasmids pSV2neo-PE, SVBss-PE, and pneoAn-PE, respectively.Cell culture, electroporation, and recombination assays. CHO cells (strain Klc) were cultured as described previously (37). Plasmid DNAs were linearized with appropriate restriction enzymes and purified by passage through a Sepharose CL-6B spin column (Pharmacia) prior to electropo...

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“…Others have suggested that nicks may improve the repair efficiency and the repair is initiated from the nick [Thomas et al 1991;Holmes et al 1990;Fang and Modrich 1993;Umar et al 1994;Miller et al 1997;Taghian et al 1998]. Conflicting studies in monkey COS-7 (CV-1 in Origin and carrying the SV40 genetic material) cells suggested that nicks did not have an effect in directing the strand repair [Heywood and Burke 1990a;1990b]. Additional studies supported this by showing equal mismatch repair efficiency with nicked and intact plasmid DNA in in vivo studies [Lei et al 2004].…”

Section: Discussionmentioning

confidence: 99%

Functional assessment for elimination of mismatches in nuclear and whole cell extracts obtained from mouse and human blastocysts

Tulay

Jaroudi

Doshi

et al. 2016

Systems Biology in Reproductive Medicine

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Preimplantation embryos may have an increased risk of having mismatches due to the rates of cell proliferation and DNA replication. Elimination of mismatches in human gametes and embryos has not been investigated. In this study we developed a sensitive functional assay to examine the repair or elimination of mismatches in both commercially available cell extracts and extracts obtained from preimplantation embryos. Heteroduplex molecules were constructed using synthetic oligonucleotides. Efficiency of the repair of mismatches was semi-quantitatively analysed by exposure to nuclear/whole cell extracts (as little as 2.5 µg) and extracts obtained from pooled mouse and human blastocysts to investigate the repair capacity in human embryos. A cell free in vitro assay was successfully developed to analyze the repair of mismatches using heteroduplex complexes. The assay was further optimized to analyze repair of mismatches in cell extracts obtained from oocytes and blastocysts using minute amounts of protein. The efficiency of mismatch repair was examined in both mouse and human blastocysts (2.5 µg). The blastocysts were observed to have a lower repair efficiency compared to commercially available nuclear and whole cell extracts. In conclusion, a sensitive, easy, and fast in vitro technique was developed to detect the repair of mismatch efficiency in embryos.

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Repair of single nucleotide DNA mismatches transfected into mammalian cells can occur by short-patch excision

Cited by 9 publications

References 21 publications

Incision at DNA G.cntdot.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.cntdot.T binding reaction

Incision at DNA G.cntdot.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.cntdot.T binding reaction

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

Functional assessment for elimination of mismatches in nuclear and whole cell extracts obtained from mouse and human blastocysts

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