TY - JOUR
T1 - Contribution of human Mlh1 and Pms2 ATPase activities to DNA mismatch repair
AU - Tomer, Guy
AU - Buermeyer, Andrew B.
AU - Nguyen, Megan M.
AU - Michael Liskay, R.
AU - Or,
PY - 2002/6/14
Y1 - 2002/6/14
N2 - MutLα, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLα and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLα proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLα demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLα activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.
AB - MutLα, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLα and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLα proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLα demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLα activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.
UR - http://www.scopus.com/inward/record.url?scp=0037077289&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037077289&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111342200
DO - 10.1074/jbc.M111342200
M3 - Article
C2 - 11897781
AN - SCOPUS:0037077289
SN - 0021-9258
VL - 277
SP - 21801
EP - 21809
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 24
ER -