Saccharomyces cerevisiae MutLalpha is a mismatch repair endonuclease

Abstract

MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLalpha (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on the integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLalpha, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLalpha is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSalpha, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLalpha endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLalpha ATPase activity or the ability of the protein to support assembly of the yMutLalpha.yMutSalpha.heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLalpha incision to the discontinuous strand of a nicked heteroduplex.

FIG. 1

Yeast MutLα is an ATP-Mn²⁺-dependent DNA endonuclease but E. coli MutL is not. (A) Alignment about the DQHA(X)₂E(X)₄E metal binding motif for human PMS2, yeast PMS1, and E. coli MutL. (B and C) Endonuclease activities of yeast MutLα (B) or E. coli MutL (C) on supercoiled homoduplex f1MR59 DNA were determined as described under “Experimental Procedures” except that ATP, MnSO₄, and MgCl₂ were varied as shown.

FIG. 2

Yeast MutLα incises the nicked strand of 3’- and 5’- heteroduplex DNA in a mismatch-, yMutSα-, yPCNA- and yRFC-dependent manner. Incision reactions (“Experimental Procedures”) contained contained proteins as indicated, nicked 3’-G-T heteroduplex (panels A and B, lanes 1-6), 3’-A•T homoduplex (lanes 7-12), nicked 5’-G-T heteroduplex DNA (panels C and D, lanes 1-6), or 5’-A•T homoduplex DNA (lanes 7-12). DNA products were cleaved with ClaI, resolved by alkaline agarose electrophoresis and transferred to nylon membranes, which were probed with ³²P-labeled oligonucleotides corresponding to viral strand coordinates 5491-5514 (A), complementary strand coordinates 2505-2526 (B and D) or viral strand coordinates 5732-5755 (C) (11,22,23). Bars with an asterisk indicate the approximate location of probe hybridization. Size markers are shown on the left. (E) Reactions containing yMutSα, yMutLα, yPCNA, yRFC, and nicked 3’-G-T heteroduplex DNA (●) or 3’-A•T homoduplex DNA (○) were performed as described above, but the KCl concentration was varied as shown. Incision of the nicked strand was quantitated by phosphorimager analysis as described previously (11) after alkaline gel electrophoresis and hybridization with a ³²P-labeled probe corresponding to viral strand coordinates 5491-5514. (F) The mismatch dependence of incision was calculated from the data in panel E.

FIG. 3

E. coli MutL, MutS, the β clamp and the γ complex do not support incision of a nicked heteroduplex DNA. (A) Reactions contained nicked 5’-G-T or 5’- A•T DNAs and proteins as indicated (“Experimental Procedures”). DNA products were resolved by electrophoresis through alkaline agarose, transferred to a nylon membrane, and probed with ³²P-labeled oligonucleotide corresponding to viral strand coordinates 5629-5652 (indicated by bar with an asterisk). Supplementation of reactions with SSB, DNA helicase II, and DNA polymerase III core did not alter the experimental outcome, and similar results were obtained with nicked 3’ heteroduplex or homoduplex DNA (not shown). (B) Left lane - reactions as in panel A were supplemented with dNTPs, SSB, DNA helicase II, exonuclease VII, and DNA polymerase III core (“Experimental Procedures”). Repair, which renders the 5’-heteroduplex sensitive to HindIII, was scored by cleavage with HindIII and ClaI. Right lane - G-T heteroduplex incubated in the absence of repair proteins was digested with HindIII and ClaI.

FIG. 4

Although defective in endonuclease activity, yeast MutLαE707K supports mismatch-dependent yMutLα•yMutSα•heteroduplex ternary complex formation. (A) Surface plasmon resonance spectroscopy was performed as described under “Experimental Procedures.” (Upper) Solutions tested contained 100 nM yMutSα (red lines), 100 nM yMutLα (blue lines), or a mixture of 100 nM of yMutSα and 100 nM yMutLα (green lines). Solid lines are results obtained with a 201 bp G-T heteroduplex, while dashed lines are those obtained with an otherwise identical A•T homoduplex. (Lower) Experimental procedures were as in the upper panel except that wild type yMutLα was replaced with yMutLαE707K. (B) Incision of nicked 3’-G-T heteroduplex DNA in the presence of yMutSα, yRFC, yPCNA, and ATP•Mg²⁺ was performed as described in “Experimental Procedures” except that yMutLα (●) and yMutLαE707K (■) concentrations were varied as indicated. DNA products were visualized and quantitated as in Fig. 2E. (C) ATP•Mn²⁺-dependent endonuclease activity on supercoiled DNA at the low ionic strength and in the absence of other proteins was determined as a function of MutLα (●) and yMutLαE707K (■) concentrations (“Experimental Procedures”).

FIG. 5

yPCNA in the presence of RFC directs yMutLα incision to a nicked DNA strand. Incision reactions containing nicked 3’-A•T homoduplex DNA were as described in “Experimental Procedures” and Fig. 2, except that yMutSα was omitted, RFC and PCNA were present as indicated, yMutLα was varied as shown, ATP concentration was 0.5 mM, 1 mM MnCl₂ was substituted for MgCl₂, and total salt concentration was 22 mM (11 mM NaCl and 11 mM KCl). Incision of the nicked (A) and continuous (B) homoduplex strands was visualized as in Fig. 2A-D. (C) Incision of the nicked (●,■) and continuous (○,□) homoduplex strands of panels A and B was quantitated as in Fig. 2E for reactions that included yMutLα, yRFC and yPCNA (●,○) or yMutLα only (■,□).