Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

Kow, Yoke W.; Bao, Gaobin; Reeves, Jason; Jinks-Robertson, Sue; Crouse, Gray F.

doi:10.1073/pnas.0704695104

Cited by 41 publications

(68 citation statements)

References 55 publications

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“…8 On the other hand, Msh6-deficiency only allowed the introduction of 1-, 2-, 3-and 4-nt substitutions, while nucleotide insertions were still suppressed by the activity of the remaining MSH2/MSH3 complex. 8 Similarly, Kow et al 50 reported that +7 nt insertions could efficiently be introduced into an msh3-deficient yeast strain, while +1 nt insertions were suppressed by residual MSH2/MSH6 activity. Conversely, msh6-deficient cells were permissive for +1 nt insertions, while +7 nt insertions were suppressed.…”

Section: Suppression By Mmr Depends On the Type Of Sequence Alterationmentioning

confidence: 97%

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

Aarts

Riele

2010

Gene Ther

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Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is a promising technique for introducing site-specific sequence alterations without affecting the genomic organization of the target locus. Here, we discuss the significant progress that has been made over the last 5 years in unraveling the mechanisms and reaction parameters underlying ssODN-mediated gene targeting. We will specifically focus on ssODN-mediated gene targeting in murine embryonic stem cells (ESCs) and the impact of the DNA mismatch repair (MMR) system on the targeting process. Implications of novel findings for routine application of ssODN-mediated gene targeting and challenges that need to be overcome for future therapeutic applications are highlighted.

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Section: Suppression By Mmr Depends On the Type Of Sequence Alterationmentioning

confidence: 97%

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

Aarts

Riele

2010

Gene Ther

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“…Our results are most consistent with a mechanism in which the oligo anneals to either the leading or lagging strand of replication at the replication fork, with subsequent extension. Mispairs created by the oligos are recognized by MMR, but those mispairs that escape MMR recognition create mutations in the next round of replication (6). For the experiments reported here, we used a mutation in an essential codon of the yeast TRP5 gene, the reversion of which occurs solely by restoration of the original codon (7).…”

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confidence: 99%

Mismatch repair-dependent mutagenesis in nondividing cells

Rodriguez¹,

Romanova²,

Bao³

et al. 2012

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

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Mismatch repair (MMR) is a major DNA repair pathway in cells from all branches of life that removes replication errors in a strandspecific manner, such that mismatched nucleotides are preferentially removed from the newly replicated strand of DNA. Here we demonstrate a role for MMR in helping create new phenotypes in nondividing cells. We show that mispairs in yeast that escape MMR during replication can later be subject to MMR activity in a replication strand-independent manner in nondividing cells, resulting in either fully wild-type or mutant DNA sequence. In one case, this activity is responsible for what appears to be adaptive mutation. This replication strand-independent MMR activity could contribute to the formation of tumors arising in nondividing cells and could also contribute to mutagenesis observed during somatic hypermutation of Ig genes.DNA mismatch repair | oligonucleotide transformation | 8-oxoG D NA mismatch repair (MMR) recognizes mismatches created in the process of replication and uses some type of strand discrimination signal to selectively remove mismatched nucleotides present on the newly replicated strand of DNA (1-3). In most eukaryotic cells, there are two mismatch recognition complexes with different, but overlapping, specificities: (i) MutSα, a heterodimer of Msh2 and Msh6, recognizing base/base mismatches and small insertion/deletion loops; and (ii) MutSβ, a heterodimer of Msh2 and Msh3, recognizing both small and large loops (1-3). After recognition of a mispair by MutSα or MutSβ, completion of MMR requires association with proteins related to MutL, usually MutLα, which in yeast is a heterodimer of Mlh1 and Pms1 (1-3). DNA on the newly synthesized strand is then excised and the template strand rereplicated.The method of strand discrimination in eukaryotic MMR is still not solved, although major advances have recently been made. Several components of MMR are known to have association with proliferating cell nuclear antigen (PCNA), a sliding clamp that tracks with the replication fork (1-3). As would be predicted by that model, it has recently been shown that MMR is temporally coupled to replication (4) and that one pathway of MutSα-dependent MMR is through recruitment by a PCNA-Msh6 interaction (5). Whatever signals are used for strand discrimination are presumably lost as replication proceeds.To study various aspects of MMR, we have used single-stranded oligonucleotides (oligos) to introduce specific mispairs into Saccharomyces cerevisiae chromosomal DNA. We have shown previously that oligos can be introduced into cells by electroporation and can correct frame-shift mutations in LYS2 and that this process is inhibited by MMR (6). Our results are most consistent with a mechanism in which the oligo anneals to either the leading or lagging strand of replication at the replication fork, with subsequent extension. Mispairs created by the oligos are recognized by MMR, but those mispairs that escape MMR recognition create mutations in the next round of replication (6). For the experiments ...

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“…Evidence suggests that ssODNs anneal to their complementary target DNA when it is transiently single-stranded during replication. However, MMR represses the efficiency of ssODN-mediated gene modification, thereby severely limiting its applicability (14,20,21). Different strategies have been reported to overcome the repressive action of MMR, which include permanent MMR disruption, transient knockdown of MMR activity, and the use of base analogs that attenuate recognition of mismatches by MMR (28)(29)(30)(31).…”

Section: Discussionmentioning

confidence: 99%

“…In mammalian, yeast, and prokaryotic cell systems, it has unequivocally been demonstrated that the DNA mismatch repair (MMR) system has a strong suppressive effect on oligo targeting efficiencies (14,20,21). Disabling the MMR system through disruption of the key MMR gene Msh2 increased the targeting efficiency up to 700-fold in mouse embryonic stem cells (mESCs) (20).…”

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confidence: 99%

LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

Ravesteyn

Dekker

Fish

et al. 2016

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

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Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.subtle gene modification | DNA mismatch repair | locked nucleic acid | single-stranded oligonucleotides | embryonic stem cells S ince the rapid decline in genome sequencing costs and the accumulation of data from genome-wide association studies, thousands of single-nucleotide variations in disease-related genes have been identified. One approach to functionally investigate these variants of uncertain clinical significance is to introduce them into the endogenous gene of appropriate model systems that are readily accessible to phenotypic assessment. Recently developed protocols for subtle gene modification are based on templatedirected repair of DNA double-strand breaks (DSBs) that are site-specifically introduced by zinc-finger, TALE, or RNA-directed Cas9 nucleases (1). In particular, subtle mutations can effectively be introduced by single-stranded DNA oligonucleotides (ssODNs) carrying the modification of interest that serve as templates for the repair of CRISPR/Cas9-introduced DSBs (2). Several laboratories, including ours, have previously shown that ssODNs can also be used for subtle gene modification in the absence of DSBs. Although less efficient than nuclease-assisted gene modification, oligonucleotide-directed gene modification (also referred to as "oligo targeting") is attractive due to its lack of additional components, simplicity, and cost-effectiveness, especially in cases where the consequences of the planned modifications can be scored by a selectable or easily detectable phenotype.Different models have been proposed for the mechanism of oligo targeting (reviewed in ref.3). Substantial evidence has been obtained that gene modification takes place during DNA replication (4-7). In this model, the ssODN anneals to singlestranded DNA in the replication fork, where it can serve as a primer for DNA synthesis by replicative polymerases (8). This process thus physically incorporates the ssODN and delivers the mutat...

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Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

Cited by 41 publications

References 55 publications

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

Mismatch repair-dependent mutagenesis in nondividing cells

LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

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