MLH1, PMS1, and MSH2 interactions during the initiation of DNA mismatch repair in yeast

Science

Volumn 265, Issue 5175, 1994, Pages 1091-1093

  Prolla, Tomas A ^a,b   Pang, Qishen ^b   Alani, Eric ^c   Kolodner, Richard D ^c   Liskay, R Michael ^b  

^a YALE UNIVERSITY   (United States)

^b OREGON HEALTH AND SCIENCE UNIVERSITY   (United States)

^c HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; BASE MISPAIRING; COMPLEX FORMATION; DNA REPAIR; NONHUMAN; PRIORITY JOURNAL; PROTEIN DNA INTERACTION; SACCHAROMYCES CEREVISIAE;

EUKARYOTA; SACCHAROMYCES CEREVISIAE;

EID: 0028128601     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.8066446     Document Type: Article

References (38)

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26. The MLH1, PMS1, and MSH2 genes were inserted into the cloning sites of plasmids pGAD (encoding GAL4 residues 768 to 881 and containing a yeast LEU2 gene as selectable marker) or pBTM (encoding the LexA DNA binding domain and containing the yeast TRP1 gene as a selectable marker) and were used to transform strains 801a or 801α (ade2 trp1-901 leu2-3, 112 his3-200 gal4 gal80 URA3::lexA op-lacZ). We generated strains carrying the desired plasmid combinations by mating haploid strains containing pGAD or pBTM plasmids and selecting for diploids in media devoid of tryptophan and leucine. Individual colonies were patched onto selective media, transferred onto nitrocellulose fitters, and grown overnight. Membranes were dropped into liquid nitrogen for 30 s, dried, and incubated at 30°C with the chromogenic substrate X-GAL [J. Breeden and K. Nasmyth, Cold Spring Harbor Symp. Quant. Biol. 50, 643 (1985)]. Color development was clearly visible after 30 min with the MLH1-PMS1 pairwise combination and a positive control, the yeast protein kinase HRR25 [M. F. Hoekstra et al, Science 253, 1031 (1991)] in combination with the HIT1 protein; the other samples were monitored over the next 12 hours. Quantitative assays revealed 5.7 U of β-galactosidase activity for the MLH1-PMS1 combination, 252 U of activity for the positive control, and less than 1 U of activity for all other combinations.
27. The MLH1 gene was subcloned into an ADE2-encoding PG-1 plasmid, which directs high-level constitutive expression in yeast [M. Shena, D. Picard, K. Yamamoto, Methods Enzymol. 194, 389 (1991)]. This construct was transformed into a diploid strain containing the pairwise combination pGAD-MSH2-pBTM-PMS1.
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29. T. A. Prolla, Q. Pang, E. Alani, R. D. Kolodner, R. M. Liskay, data not shown.
30. 32P]ATP. For gel retardation assays, MLH1, PMS1, and MSH2 were initially prepared as MBP fusion proteins and were eluted from amylose resin with column buffer containing 10 mM maltose. Factor Xa (New England Biolabs) was added to a final concentration of 20 μml, and the mixture was incubated at 4°C for 16 hours. The fusion protein cleavage mixture was absorbed on a hydroxyapatite column, and the maltose-free proteins were eluted with 0.5 M sodium phosphate. Proteins were then passed through a second amylose column. The proteins in the flow-through fractions were found to be mostly free of the MBP domain, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were stored at -20°C.
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38. We thank M. G. Marinus for plasmids pPY97 and pMQ269, M. Hoekstra for the HRR25 and HIT1 positive control plasmids, and M. Thayer and M. Hoekstra for critical comments on the manuscript. Supported by an NSF graduate fellowship (T.A.P.), a Merck fellowship from the Life Sciences Research Foundation (E.A.), NSF grant MCB 9314116 and NIH grants GM 45413 and GM 322741 (R.M.L.), and NIH grant HG00305/ GM50006 (R.D.K.).