PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair

Abstract

MutLα (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion.

Fig. 1.

RFC function in MutLα activation is limited to PCNA loading. (A) Human PCNA was loaded onto 3′ G-T heteroduplex DNA using yRFC∆N, and reaction products subjected to Superdex 200 gel filtration (Materials and Methods). DNA eluted in fractions 15–18 and unbound protein in fractions 25–31. (B) The yRFC∆N dependence of MutLα endonuclease activation was determined using 24 fmol 3′ G–T heteroduplex DNA as described in Materials and Methods. Values shown are corrected for a modest background level of random strand breaks in the heteroduplex preparation used. The bracket indicates the activity range supported by the yRFC∆N levels present in 10 μL of Superdex 200 fractions 15–18 as estimated by Western blot (Fig. S2B). (C) Ten μL of the indicated Superdex 200 fractions were assayed for strand-directed endonuclease activity in the absence (red bars) or presence of MutSα and MutLα [blue bars (Materials and Methods), mismatch-dependent conditions)]. Background strand breaks in the heteroduplex preparation used were comparable to those observed when MutSα and MutLα were omitted from the incubation (red bars). Raw data for this analysis is shown in Fig. S2A.

Fig. 2.

Endonuclease activity on linear DNA requires PCNA but not RFC. Mismatch-independent MutLα endonuclease activity on 202 bp linear homoduplex DNA as described in Materials and Methods in the presence of reaction components as indicated. (A) Reactions contained 30 mM KCl, 0.5 mM ATP, 1 mM MnSO₄, and protein components as indicated. (B) Reactions contained 60 mM KCl, 1.5 mM ATP, 5 mM MgCl₂, and indicated proteins. ATP was omitted from the reaction in lane 8 and EDTA substituted for MgCl₂ in lane 9. Reactions shown in lanes 11–13 were performed as for lane 8 except that final KCl concentration was varied as shown.

Fig. 3.

Strand break and DNA topology dependence of PCNA loading and MutLα activation. Efficiency of PCNA loading onto nicked, supercoiled (σ = -0.12), relaxed bubble, and relaxed G–T heteroduplexes was determined by gel filtration assay (Materials and Methods and Fig. S4). Loading values shown are corrected for that observed in the absence of ATP (0.5 PCNA trimer/DNA). Clamp loading utilized native human RFC (A) or yRFCΔN (B). For supercoiled DNA, loading values are the mean of 3 determinations (± 1 standard deviation); for other DNAs, values are the average of 2 determinations, with the variation shown corresponding to the range observed. MutLα endonucleolytic incision of complementary (C) and viral (V) strands of these heteroduplexes (or A–T homoduplex controls) was determined in the presence of the indicated proteins and quantified as described under Materials and Methods, and corrected for presence of background nicks in DNA preparations as described in Fig. 1. Incision values indicated with an asterisk are the average of 2 determinations, with the range observed indicated. Other values are the mean of 3 to 6 determinations ± one standard deviation.

Fig. 4.

MutLα activation as a function of extent of PCNA loading. Extents of MutLα activation are plotted against efficiencies of PCNA loading by RFC and yRFCΔN for the heteroduplexes shown in Fig. 3. Lines shown are empirical fits.

Fig. 5.

Model for PCNA involvement in the strand direction of MutLα endonuclease. As indicated by blue and tan colors, the two faces of PCNA are nonequivalent, and the clamp is loaded with a unique orientation relative to the 3′ double strand-single strand junction at a nick or gap (A). A transient (22) or artificial bubble (this study) within a covalently closed circular DNA also supports clamp loading. Because the double strand-single strand junctions at the two ends of a bubble are rotationally symmetric, such structures must support PCNA loading in either orientation (B). We suggest that the MutLα–PCNA complex is characterized by intrinsic asymmetry and that this asymmetry coupled with PCNA loading orientation dictates the strand specificity of incision by the MutLα endonuclease active site within the PMS2 subunit.