Leonid Dzantiev

Endonucleolytic Function of MutLα in Human Mismatch Repair

Summary Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLα (MLH1•PMS2 heterodimer). MutLα is required for mismatch repair, but its function in this process is unclear. We show that human MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5′-to-3′ activity of MutSα-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X) 2 E(X) 4 E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.

Human mismatch repair: reconstitution of a nick-directed bidirectional reaction.

Bidirectional mismatch repair directed by a strand break located 3′ or 5′ to the mispair has been reconstituted using seven purified human activities: MutSα, MutLα, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase δ. In addition to DNA polymerase δ, PCNA, RFC, and RPA, 5′-directed repair depends on MutSα and EXOI, whereas 3′-directed mismatch correction also requires MutLα. The repair reaction displays specificity for DNA polymerase δ, an effect that presumably reflects interactions with other repair activities. Because previous studies have suggested potential involvement of the editing function of a replicative polymerase in mismatch-provoked excision, we have evaluated possible participation of DNA polymerase δ in the excision step of repair. RFC and PCNA dramatically activate polymerase δ-mediated hydrolysis of a primer-template. Nevertheless, the contribution of the polymerase to mismatch-provoked excision is very limited, both in the purified system and in HeLa extracts, as judged by in vitro assay using nicked circular heteroplex DNAs. Thus, excision and repair in the purified system containing polymerase δ are reduced 10-fold upon omission of EXOI or by substitution of a catalytically dead form of the exonuclease. Furthermore, aphidicolin inhibits both 3′- and 5′-directed excision in HeLa nuclear extracts by only 20–30%. Although this modest inhibition could be because of nonspecific effects, it may indicate limited dependence of bidirectional excision on an aphidicolin-sensitive DNA polymerase.

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

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.

The MutSalpha-proliferating cell nuclear antigen interaction in human DNA mismatch repair.

We have examined the interaction parameters, conformation, and functional significance of the human MutSα· proliferating cell nuclear antigen (PCNA) complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a KD of 0.7 μm in the absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSα for a mismatch, and mismatch-bound MutSα binds PCNA. Small angle x-ray scattering studies have established the molecular parameters of the complex, which are consistent with an elongated conformation in which the two proteins associate in an end-to-end fashion in a manner that does not involve an extended unstructured tether, as has been proposed for yeast MutSα and PCNA ( Shell, S. S., Putnam, C. D., and Kolodner, R. D. (2007) Mol. Cell 26, 565-578 ). MutSα variants lacking the PCNA interaction motif are functional in 3′- or 5′-directed mismatch-provoked excision, but display a partial defect in 5′-directed mismatch repair. This finding is consistent with the modest mutability conferred by inactivation of the MutSα PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in MutSα-dependent mismatch repair.

Interaction of Escherichia coli DNA polymerase I (Klenow fragment) with primer-templates containing N-acetyl-2-aminofluorene or N-2-aminofluorene adducts in the active site

DNA adducts formed by aromatic amines such asN-acetyl-2-aminofluorene (AAF) andN-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication. To understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primer-templates modified with an AAF or AF adduct. The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA. More specifically, it was found that the binding is 5–10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site. In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide. The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower. In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present.