Mismatch-provoked Excision Research Articles

The Effects of Galactic Cosmic Radiation Exposure on Hematopoietic Stem Cell Dysfunction and Oncogenesis

Natural sources of radiation in space include galactic cosmic rays (GCR), solar energetic particles (SPE) and trapped energetic particles in a planetary magnetic field. These different sources of space radiation consist of protons of various energies, particle nuclei of high energy and charge (HZE) and neutrons of different energies. These sources are difficult to shield because of their high energies and dense ionization patterns, thus posing significant health risks to astronauts on long term inter-planetary missions. Efforts to protect astronauts from harmful cosmic radiation require a deeper understanding of the effects of GCR on human health. In particular, very little is known about the effects of GCR exposure on the hematopoietic stem cell (HSC) population and whether disruptions in genetic stability in HSCs could result in the development of hematopoietic malignancies in astronauts on deep space missions.The average age of shuttle crew has risen above 46 years, and our work and others have shown that HSCs display diminished function with age. Recent data from our group has demonstrated that middle-aged individuals show frequent defects in DNA mismatch repair (MMR) in HSCs. MMR corrects DNA mismatches generated by DNA polymerase during replication which prevents mutations from becoming permanent in dividing cells. Thus, MMR plays a crucial role in the DNA damage response pathway to prevent short-term mutagenesis and long-term tumorigenesis. Several human MMR proteins have been identified as MutS and MutL homologues consisting of MSH2 and MLH1 heterodimers that functions in DNA mismatch/damage recognition, endonuclease activity and termination of mismatch-provoked excision. Our group has shown that humans accumulate microsatellite instability (MSI) with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age. Therefore, we employed a DNA mismatch repair deficient mouse model (MLH1+/- and MLH1-/-) to study the effects of different radiation sources including 56Fe, 28Si, 4He, 1H and γ-rays on HSCs to examine HSCs of potential astronaut population under GCR conditions. The complete blood count (CBC) data after 5 months and 9 months of whole body irradiation with different ions showed a slight dose-dependent decrease in all blood counts but absence of any significant difference in CBC of MLH1+/+ vs MLH1+/- mice. In addition, CFU and competitive repopulation data demonstrated a radiation quality effects on HSC function, but not an MLH1 effect. These results demonstrate that hematopoietic stem cell function is normal and that a MLH1 defect does not differentiate progenitor and mature effector cells following HZE radiation.To study long term effects of different ions on the potential for disease progression in a MLH1 dependent manner, we performed whole body irradiation with 56Fe, 28Si, 1H and γ-rays on MLH1+/+ and MLH1+/- mice and followed them up to 18 months post exposure. We observed that MLH1+/- mice show dramatic increases in lymphomagenesis 10-12 months after 56Fe irradiation compared to wild type mice, with greater than 60 % of MLH1+/- mice developed lymphomas at doses 10 cGy and 100 cGy compared to less than 10 % of wild type. For comparison, roughly 10 % of MLH1+/- mice developed lymphomas when mice were treated with whole body sparsely ionizing γ-rays at 100 cGy compared to none of the control. Thus the date show that MMR defects in HSCs lead to sensitization to radiation induced hematopoietic malignancy and that radiation quality effects exacerbate the sensitivity. The findings could have profound effects on astronaut screening, as well as lead to important questions regarding safety of ion therapy and development of second malignancies for cancer patients who remain on Earth. DisclosuresNo relevant conflicts of interest to declare.

Hydrolytic function of Exo1 in mammalian mismatch repair.

Genetic and biochemical studies have previously implicated exonuclease 1 (Exo1) in yeast and mammalian mismatch repair, with results suggesting that function of the protein in the reaction depends on both its hydrolytic activity and its ability to interact with other components of the repair system. However, recent analysis of an Exo1-E109K knockin mouse has concluded that Exo1 function in mammalian mismatch repair is restricted to a structural role, a conclusion based on a prior report that N-terminal His-tagged Exo1-E109K is hydrolytically defective. Because Glu-109 is distant from the nuclease hydrolytic center, we have compared the activity of untagged full-length Exo1-E109K with that of wild type Exo1 and the hydrolytically defective active site mutant Exo1-D173A. We show that the activity of Exo1-E109K is comparable to that of wild type enzyme in a conventional exonuclease assay and that in contrast to a D173A active site mutant, Exo1-E109K is fully functional in mismatch-provoked excision and repair. We conclude that the catalytic function of Exo1 is required for its participation in mismatch repair. We also consider the other phenotypes of the Exo1-E109K mouse in the context of Exo1 hydrolytic function.

Termination of exonuclease 1‐catalyzed mismatch excision requires physical interaction between exonuclease 1 and MutLα

The human mismatch repair (MMR) reaction was previously reconstituted in vitro using purified recombinant proteins that include MutSα or MutSβ, MutLα, RPA, EXO1, HMGB1, PCNA, RFC, polymerase δ, and DNA ligase I. Although MutLα is not essential for 5′ nick‐directed excision, it promotes termination of mismatch‐provoked EXO1‐ catalyzed DNA excision upon mismatch removal. However, the mechanism of the excision termination reaction is unknown. This study examines whether the physical interaction between the MLH1 subunit of MutLα and EXOI, which is mediated by a conserved motif in EXO1 known as an MLH1 interacting protein (MIP)‐box, is responsible for the excision termination. For this purpose, alanine substitution mutations were engineered at residues F506 and F507 in the MIP‐box of EXO1, and the resulting FF‐EXO‐AA mutant protein was tested for its role in MMR in vitro. As expected, FF‐EXO1‐AA behaves similarly as wild type EXO1 in catalyzing 5′‐directed excision and interacting with MutSα, but the EXO1 mutant protein fails to interact with MutLα. Our in vitro MMR assay show that mismatch‐provoked excision is efficiently terminated in reactions containing wild type EXO1, but not in reactions containing FF‐EXO1‐ AA. These observations strongly suggest that the termination of EXO1‐catalyzed mismatch excision is executed via physical interaction between MutLα and EXO1.

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

PARP-1 enhances the mismatch-dependence of 5′-directed excision in human mismatch repair in vitro

End-directed mismatch-provoked excision has been reconstituted in several purified systems. While 3′-directed excision displays a mismatch dependence similar to that observed in nuclear extracts (≈20-fold), the mismatch dependence of 5′-directed excision is only 3–4-fold, significantly less than that in extracts (8–10-fold). Utilizing a fractionation-based approach, we have isolated a single polypeptide that enhances mismatch dependence of reconstituted 5′-directed excision and have shown it to be identical to poly[ADP-ribose] polymerase 1 (PARP-1). Titration of reconstituted excision reactions or PARP-1-depleted HeLa nuclear extract with purified PARP-1 showed that the protein specifically enhances mismatch dependence of 5′-directed excision. Analysis of a set of PARP-1 mutants revealed that the DNA binding domain and BRCT fold contribute to the regulation of excision specificity. Involvement of the catalytic domain is restricted to its ability to poly(ADP-ribosyl)ate PARP-1 in the presence of NAD +, likely through interference with DNA binding. Analysis of protein-protein interactions demonstrated that PARP-1 interacts with mismatch repair proteins MutSα, exonuclease 1, replication protein A (RPA), and as previously shown by others, replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) as well. The BRCT fold plays an important role in the interaction of PARP-1 with the former three proteins.

MutLα and Proliferating Cell Nuclear Antigen Share Binding Sites on MutSβ

MutSβ (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSβ interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSβ-PCNA complex formation occurs with an affinity of ∼0.1 μm and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSβ is in molar excess over PCNA. Conformational studies indicate that the two proteins associate in an end-to-end fashion in solution. Surprisingly, mutation of the PCNA-binding motif of MutSβ not only abolishes PCNA binding, but unlike MutSα, also dramatically attenuates MutSβ-MutLα interaction, MutLα endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLα for binding to MutSβ, an effect that is blocked by the cell cycle regulator p21CIP1. We propose that MutSβ-MutLα interaction is mediated in part by residues ((L/I)SRFF) embedded within the MSH3 PCNA-binding motif. To our knowledge this is the first case where residues important for PCNA binding also mediate interaction with a second protein. These findings also indicate that MutSβ- and MutSα-initiated repair events differ in fundamental ways.

Involvement of the β Clamp in Methyl-directed Mismatch Repair in Vitro

We have examined function of the bacterial β replication clamp in the different steps of methyl-directed DNA mismatch repair. The mismatch-, MutS-, and MutL-dependent activation of MutH is unaffected by the presence or orientation of loaded β clamp on either 3′ or 5′ heteroduplexes. Similarly, β is not required for 3′ or 5′ mismatch-provoked excision when scored in the presence of γ complex or in the presence of γ complex and DNA polymerase III core components. However, mismatch repair does not occur in the absence of β, an effect we attribute to a requirement for the clamp in the repair DNA synthesis step of the reaction. We have confirmed previous findings that β clamp interacts specifically with MutS and MutL (López de Saro, F. J., Marinus, M. G., Modrich, P., and O'Donnell, M. (2006) J. Biol. Chem. 281, 14340–14349) and show that the mutator phenotype conferred by amino acid substitution within the MutS N-terminal β-interaction motif is the probable result of instability coupled with reduced activity in multiple steps of the repair reaction. In addition, we have found that the DNA polymerase III α catalytic subunit interacts strongly and specifically with both MutS and MutL. Because interactions of polymerase III holoenzyme components with MutS and MutL appear to be of limited import during the initiation and excision steps of mismatch correction, we suggest that their significance might lie in the control of replication fork events in response to the sensing of DNA lesions by the repair system.

Functions of MutLα, Replication Protein A (RPA), and HMGB1 in 5′-Directed Mismatch Repair

A purified system comprised of MutSα, MutLα, exonuclease 1 (Exo1), and replication protein A (RPA) (in the absence or presence of HMGB1) supports 5′-directed mismatch-provoked excision that terminates after mismatch removal. MutLα is not essential for this reaction but enhances excision termination, although the basis of this effect has been uncertain. One model attributes the primary termination function in this system to RPA, with MutLα functioning in a secondary capacity by suppressing Exo1 hydrolysis of mismatch-free DNA (Genschel, J., and Modrich, P. (2003) Mol. Cell 12, 1077–1086). A second invokes MutLα as the primary effector of excision termination (Zhang, Y., Yuan, F., Presnell, S. R., Tian, K., Gao, Y., Tomkinson, A. E., Gu, L., and Li, G. M. (2005) Cell 122, 693–705). In the latter model, RPA provides a secondary termination function, but together with HMGB1, also participates in earlier steps of the reaction. To distinguish between these models, we have reanalyzed the functions of MutLα, RPA, and HMGB1 in 5′-directed mismatch-provoked excision using purified components as well as mammalian cell extracts. Analysis of extracts derived from A2780/AD cells, which are devoid of MutLα but nevertheless support 5′-directed mismatch repair, has demonstrated that 5′-directed excision terminates normally in the absence of MutLα. Experiments using purified components confirm a primary role for RPA in terminating excision by MutSα-activated Exo1 but are inconsistent with direct participation of MutLα in this process. While HMGB1 attenuates excision by activated Exo1, this effect is distinct from that mediated by RPA. Assay of extracts derived from HMGB1+/+ and HMGB1−/− mouse embryo fibroblast cells indicates that HMGB1 is not essential for mismatch repair.

The MutSα-Proliferating Cell Nuclear Antigen Interaction in Human DNA Mismatch Repair

We have examined the interaction parameters, conformation, and functional significance of the human MutSalpha(.) proliferating cell nuclear antigen (PCNA) complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a K(D) of 0.7 microm in the absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSalpha for a mismatch, and mismatch-bound MutSalpha 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 MutSalpha and PCNA ( Shell, S. S., Putnam, C. D., and Kolodner, R. D. (2007) Mol. Cell 26, 565-578 ). MutSalpha 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 MutSalpha PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in MutSalpha-dependent mismatch repair.

Regulation of Replication Protein A Functions in DNA Mismatch Repair by Phosphorylation

Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase delta-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR.

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: MutSalpha, MutLalpha, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase delta. In addition to DNA polymerase delta, PCNA, RFC, and RPA, 5'-directed repair depends on MutSalpha and EXOI, whereas 3'-directed mismatch correction also requires MutLalpha. The repair reaction displays specificity for DNA polymerase delta, 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 delta in the excision step of repair. RFC and PCNA dramatically activate polymerase delta-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 delta 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.

Reconstitution of 5′-Directed Human Mismatch Repair in a Purified System

This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.

Is hEXO1 a Cancer Predisposing Gene?

Is <i>hEXO1</i> a Cancer Predisposing Gene?

A Defined Human System That Supports Bidirectional Mismatch-Provoked Excision

Mismatch-provoked excision directed by a strand break located 3′ or 5′ to the mispair has been reconstituted using purified human proteins. While MutSα, EXOI, and RPA are sufficient to support hydrolysis directed by a 5′ strand break, 3′ directed excision also requires MutLα, PCNA, and RFC. EXOI interacts with PCNA. RFC and PCNA suppress EXOI-mediated 5′ to 3′ hydrolysis when the nick that directs excision is located 3′ to the mispair and activate 3′ to 5′ excision, which is dependent on loaded PCNA and apparently mediated by a cryptic EXOI 3′ to 5′ hydrolytic function. By contrast, RFC and PCNA have only a limited effect on 5′ to 3′ excision directed by a 5′ strand break.

Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A.

DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.

Human Exonuclease I Is Required for 5′ and 3′ Mismatch Repair

We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted extracts requires both hMutSalpha and hMutLalpha. However, excision directed by a 5' strand break requires hMutSalpha but can occur in absence of hMutLalpha. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.

DNA-dependent Activation of the hMutSα ATPase

ATP hydrolysis by MutS homologs is required for function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is controversial. In this paper we describe a steady-state kinetic analysis of the DNA-activated ATPase of human MutSalpha. Comparison of salt concentration effects on mismatch repair and mismatch-provoked excision in HeLa nuclear extracts with salt effects on the DNA-activated ATPase suggests that ATP hydrolysis by MutSalpha is involved in the rate determining step in the repair pathway. While the ATPase is activated by homoduplex and heteroduplex DNA, the half-maximal concentration for activation by heteroduplex DNA is significantly lower under physiological salt concentrations. Furthermore, at optimal salt concentration, heteroduplex DNA increases the kcat for ATP hydrolysis to a greater extent than does homoduplex DNA. We also demonstrate that the degree of ATPase activation is dependent on DNA chain length, with the kcat for hydrolysis increasing significantly with chain length of the DNA cofactor. These results are discussed in terms of the translocation (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997) EMBO J. 16, 4467-4476) and the molecular switch (Gradia, S., Acharya, S., and Fishel, R. (1997) Cell 91, 995-1005) models that invoke distinct roles for ATP hydrolysis in MutS homolog function.

MutS and MutL Activate DNA Helicase II in a Mismatch-dependent Manner

MutS, MutL, and DNA helicase II are required for the mismatch-provoked excision step that occurs during Escherichia coli methyl-directed mismatch repair. In this study MutL is shown to enhance the unwinding activity of DNA helicase II more than 10-fold on a conventional helicase substrate in which a 35-residue oligonucleotide is annealed to a M13 circular single-stranded phage DNA under conditions where the two proteins are present at approximately molar stoichiometry with respect to the substrate. MutS- and MutL-dependent activation of DNA helicase II has also been demonstrated with a model substrate in which a 138-residue oligonucleotide was hybridized to a 138-nucleotide gap in an otherwise duplex 7,100-base pair circular DNA. Displacement of the oligonucleotide requires MutS, MutL, DNA helicase II, and ATP and is dependent on the presence of a mismatch within the hybrid region. Although DNA helicase II and Rep helicase share substantial sequence homology and features of mechanism, Rep helicase is inactive in this reaction.

Bidirectional excision in methyl-directed mismatch repair

Using electron microscopy and indirect end-labeling methods, we have examined excision tracts produced by the Escherichia coli methyl-directed mismatch repair system on a closed circular G-T heteroduplex that contains a single d(GATC) site. Despite differing polarities of the unmodified strand in the two hemimethylated derivatives of the heteroduplex, that portion of the unmethylated strand spanning the shorter path between the d(GATC) site and mismatch is targeted for excision in both cases. Mismatch-provoked excision occurring on both hemimethylated DNAs requires DNA helicase II, but exonuclease requirements for the reaction depend on heteroduplex orientation. When the d(GATC) sequence on the unmodified strand resides 3' to the mismatch as viewed along the shorter path, excision requires exonuclease I. Excision occurring on the alternate hemimethylated heteroduplex depends on the 5'--> 3' hydrolytic activity of exonuclease VII. Coupled with the previous demonstration that repair initiates via the mismatch-provoked, MutHLS-dependent incision of the unmethylated strand at a d(GATC) sequence (Au, K.G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148), these findings indicate an excision mechanism in which helicase II displacement renders the incised strand sensitive to the appropriate single-strand exonuclease. Our data imply that hydrolysis commences at the d(GATC) site, proceeds to a point beyond the mismatch, and terminates at a number of discrete sites within a 100-nucleotide region just beyond this site. The extent of excision is therefore controlled by one or more components of the repair system.