| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Papers In Press, published online ahead of print May 10, 2002
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331
Corresponding Author: haysj{at}bcc.orst.edu
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 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 preexisting 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 preexisting 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.
J. Biol. Chem, 10.1074/jbc.M200357200
Submitted on January 11, 2002
Revised on May 8, 2002
Accepted on May 9, 2002
Mismatch repair in human nuclear extracts: Quantitative kinetic analyses of excision of nicked circular mismatched DNA substrates, constructed by a new technique employing synthetic oligonucleotides
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Wang and J. B. Hays Human DNA mismatch repair: coupling of mismatch recognition to strand-specific excision Nucleic Acids Res., November 29, 2007; 35(20): 6727 - 6739. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. W. Hou, R. Prasad, K. Asagoshi, A. Masaoka, and S. H. Wilson Comparative assessment of plasmid and oligonucleotide DNA substrates in measurement of in vitro base excision repair activity Nucleic Acids Res., September 27, 2007; 35(17): e112 - e112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-Y. Xu, Z. Zhu, P. Zhang, S.-H. Chan, J. C. Samuelson, J. Xiao, D. Ingalls, and G. G. Wilson Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI Nucleic Acids Res., July 9, 2007; 35(14): 4608 - 4618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Plotz, C. Welsch, L. Giron-Monzon, P. Friedhoff, M. Albrecht, A. Piiper, R. M. Biondi, T. Lengauer, S. Zeuzem, and J. Raedle Mutations in the MutS{alpha} interaction interface of MLH1 can abolish DNA mismatch repair Nucleic Acids Res., December 2, 2006; 34(22): 6574 - 6586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Modrich Mechanisms in Eukaryotic Mismatch Repair J. Biol. Chem., October 13, 2006; 281(41): 30305 - 30309. [Full Text] [PDF]
|
|
|
|
|
|
S. J. York and P. Modrich Mismatch Repair-dependent Iterative Excision at Irreparable O6-Methylguanine Lesions in Human Nuclear Extracts J. Biol. Chem., August 11, 2006; 281(32): 22674 - 22683. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Karnitz, K. S. Flatten, J. M. Wagner, D. Loegering, J. S. Hackbarth, S. J. H. Arlander, B. T. Vroman, M. B. Thomas, Y.-U. Baek, K. M. Hopkins, et al. Gemcitabine-Induced Activation of Checkpoint Signaling Pathways That Affect Tumor Cell Survival Mol. Pharmacol., December 1, 2005; 68(6): 1636 - 1644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yang, L. E. Sass, C. Du, P. Hsieh, and D. A. Erie Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions Nucleic Acids Res., August 1, 2005; 33(13): 4322 - 4334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kang, S. Huang, and M. J. Blaser Structural and Functional Divergence of MutS2 from Bacterial MutS1 and Eukaryotic MSH4-MSH5 Homologs J. Bacteriol., May 15, 2005; 187(10): 3528 - 3537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-M. Huang, S.-U. Chen, S. D. Goodman, S.-H. Wu, J.-T. Kao, C.-N. Lee, W.-C. Cheng, K.-S. Tsai, and W.-h. Fang Interaction of Nick-directed DNA Mismatch Repair and Loop Repair in Human Cells J. Biol. Chem., July 16, 2004; 279(29): 30228 - 30235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Martik, C. Baitinger, and P. Modrich Differential Specificities and Simultaneous Occupancy of Human MutS{alpha} Nucleotide Binding Sites J. Biol. Chem., July 2, 2004; 279(27): 28402 - 28410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang and J. B. Hays Mismatch Repair in Human Nuclear Extracts: EFFECTS OF INTERNAL DNA-HAIRPIN STRUCTURES BETWEEN MISMATCHES AND EXCISION-INITIATION NICKS ON MISMATCH CORRECTION AND MISMATCH-PROVOKED EXCISION J. Biol. Chem., August 1, 2003; 278(31): 28686 - 28693. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang and J. B. Hays Mismatch Repair in Human Nuclear Extracts. TIME COURSES AND ATP REQUIREMENTS FOR KINETICALLY DISTINGUISHABLE STEPS LEADING TO TIGHTLY CONTROLLED 5' TO 3' AND APHIDICOLIN-SENSITIVE 3' TO 5' MISPAIR-PROVOKED EXCISION J. Biol. Chem., July 12, 2002; 277(29): 26143 - 26148. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
