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

2016 ◽  
Vol 113 (15) ◽  
pp. 4122-4127 ◽  
Author(s):  
Thomas W. van Ravesteyn ◽  
Marleen Dekker ◽  
Alexander Fish ◽  
Titia K. Sixma ◽  
Astrid Wolters ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Embryonic Stem ◽  
Gene Modification ◽  
Single Stranded Dna ◽  
Dna Mismatch ◽  
Dna Oligonucleotides ◽  
Single Base Pair Substitution ◽  
Genetic Modifications ◽  
Single Base Pair

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.

Gene Modification in Embryonic Stem Cells by Single-Stranded DNA Oligonucleotides

2009 ◽  
pp. 79-99 ◽  
Author(s):  
Marieke Aarts ◽  
Marleen Dekker ◽  
Rob Dekker ◽  
Sandra de Vries ◽  
Anja van der Wal ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Gene Modification ◽  
Single Stranded Dna ◽  
Dna Oligonucleotides

Repair of single base-pair transversion mismatches of Escherichia coli in vitro: correction of certain A/G mismatches is independent of dam methylation and host mutHLS gene functions.

Genetics ◽  
1988 ◽  
Vol 118 (4) ◽  
pp. 593-600 ◽  
Author(s):  
A L Lu ◽  
D Y Chang
Keyword(s):  
Mismatch Repair ◽  
Base Pair ◽  
Repair System ◽  
Gene Products ◽  
Dam Methylation ◽  
Gene Functions ◽  
Mismatch Repair System ◽  
Single Base Pair ◽  
Repair Pathways

Abstract Six different base-pair transversion mismatches are repaired with different efficiencies in an in vitro mismatch repair system. In particular, the T/T and C/C mismatches appear to be less efficiently repaired than the A/A and G/G mismatches. Four A/G and four C/T mismatches at different positions are repaired to different extents. One of the A/G mismatches is repaired equally efficiently when DNA heteroduplexes are fully methylated or hemi-methylated at the d(GATC) sequences. This type of mismatch repair appears to be unidirectional with A to C conversion by acting at A/G mispairs to restore the C/G pairs. This methylation-independent correction is not controlled by the mutH, mutL, mutS, uvrE, uvrB, phr, recA, recF, and recJ gene products. The independence of the transversion mismatch repair of these genes and methylation distinguishes this from the known mismatch repair pathways.

scholarly journals Mismatch Repair Processing of Carcinogen-DNA Adducts Triggers Apoptosis

1999 ◽  
Vol 19 (12) ◽  
pp. 8292-8301 ◽  
Author(s):  
Jianxin Wu ◽  
Liya Gu ◽  
Huixian Wang ◽  
Nicholas E. Geacintov ◽  
Guo-Min Li
Keyword(s):  
Dna Damage ◽  
Mismatch Repair ◽  
Repair System ◽  
Apoptotic Response ◽  
Chemical Carcinogens ◽  
P53 Expression ◽  
Dna Mismatch ◽  
Repair Deficiency ◽  
Mismatch Repair System

ABSTRACT The DNA mismatch repair pathway is well known for its role in correcting biosynthetic errors of DNA replication. We report here a novel role for mismatch repair in signaling programmed cell death in response to DNA damage induced by chemical carcinogens. Cells proficient in mismatch repair were highly sensitive to the cytotoxic effects of chemical carcinogens, while cells defective in either human MutS or MutL homologs were relatively insensitive. Since wild-type cells but not mutant cells underwent apoptosis upon treatment with chemical carcinogens, the apoptotic response is dependent on a functional mismatch repair system. By analyzing p53 expression in several pairs of cell lines, we found that the mismatch repair-dependent apoptotic response was mediated through both p53-dependent and p53-independent pathways. In vitro biochemical studies demonstrated that the human mismatch recognition proteins hMutSα and hMutSβ efficiently recognized DNA damage induced by chemical carcinogens, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. Taken together, these studies further elucidate the mechanism by which mismatch repair deficiency predisposes to cancer, i.e., the deficiency not only causes a failure to repair mismatches generated during DNA metabolism but also fails to direct damaged and mutation-prone cells to commit suicide.

scholarly journals Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

2017 ◽  
Vol 45 (16) ◽  
pp. 9427-9440 ◽  
Author(s):  
Dekang Liu ◽  
Jane H. Frederiksen ◽  
Sascha E. Liberti ◽  
Anne Lützen ◽  
Guido Keijzers ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Double Mutant ◽  
Dna Mismatch Repair ◽  
Dna Polymerase Delta ◽  
Dna Mismatch ◽  
Human Dna

scholarly journals In vitro studies of DNA mismatch repair proteins

2011 ◽  
Vol 413 (2) ◽  
pp. 179-184 ◽  
Author(s):  
Hui Geng ◽  
Chunwei Du ◽  
Siying Chen ◽  
Vincenzo Salerno ◽  
Candela Manfredi ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
In Vitro Studies ◽  
Dna Mismatch ◽  
Mismatch Repair Proteins
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