Abstract
Mammalian mismatch repair (MMR) systems respond to broad ranges of DNA mismatches and lesions. Kinetic analyses of MMR processing in vitro have focused on base mismatches in a few sequence contexts, because of a lack of general and quantitative MMR assays and because of the difficulty of constructing a multiplicity of MMR substrates, particularly those with DNA lesions. We describe here simple and efficient construction of 11 different MMR substrates, by ligating synthetic oligomers into gapped plasmids generated using sequence-specific N.BstNBI nicking endonuclease, then using sequence-specific nicking endonuclease N.AlwI to introduce single nicks for initiation of 3' to 5' or 5' to 3' excision. To quantitatively assay MMR excision gaps in base-mispaired substrates, generated in human nuclear extracts lacking exogenous dNTPs, we used position- and strand-specific oligomer probes. Mispair-provoked excision along the shorter path from the pre-existing nick toward the mismatch, either 3' to 5' or 5' to 3', predominated over longer path excision by roughly 10:1 to 20:1. MMR excision was complete within 7 min, was highly specific (90:1) for the nicked strand, and was strongly mispair-dependent (at least 40:1). Nonspecific (mismatch-independent) 5' to 3' excision was considerably greater than nonspecific 3' to 5' excision, especially at pre-existing gaps, but was not processive. These techniques can be used to construct and analyze MMR substrates with DNA mismatches or lesions in any sequence context.
Highlights
Mismatch repair (MMR)1 protein systems were originally defined by studies that elucidated roles of MutS-homolog (MSH) and MutL-homolog (MLH/PMS) proteins in DNA replication and recombination fidelity [1,2,3]
The mismatch repair (MMR) excision assay procedures are applicable to DNA mismatches or lesions in any sequence context
We describe here a simple and general technique to obtain, in high yield and purity, a wide variety of DNA substrates for analysis of MMR processing
Summary
QUANTITATIVE ANALYSES OF EXCISION OF NICKED CIRCULAR MISMATCHED DNA SUBSTRATES, CONSTRUCTED BY A NEW TECHNIQUE EMPLOYING SYNTHETIC OLIGONUCLEOTIDES*. The elevated UV mutagenesis seen in MMR-deficient bacterial [17] and rodent cells [18] suggests that MMR processing might correct photoproduct/base mispairs Beyond such binding experiments, exogenous circular DNA substrates containing specific base mismatches and defined nicks for initiation of excision have been used to analyze MMR error corrections in mammalian cell-free extract [19, 20]. Exogenous circular DNA substrates containing specific base mismatches and defined nicks for initiation of excision have been used to analyze MMR error corrections in mammalian cell-free extract [19, 20] These studies have yielded important mechanistic findings, including roles for MutL-homolog proteins, and for other MMR accessory proteins such as proliferating cell nuclear antigen, and demonstration of MMR excision specificity. The MMR excision assay procedures are applicable to DNA mismatches or lesions in any sequence context
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