hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress.

Genotoxic stress activates the phosphatidylinositol 3-kinase-like kinases (PIKKs) that phosphorylate proteins involved in cell cycle arrest, DNA repair and apoptosis. Previous work showed that the PIKK ataxia telangiectasia mutated (ATM) but not ATM and Rad3 related phosphorylates p53 (Ser15) during hyperoxia, a model of prolonged oxidative stress and DNA damage. Here, we show hSMG-1 is responsible for the rapid and early phosphorylation of p53 (Ser15) and that ATM helps maintain phosphorylation after 24 h. Despite reduced p53 phosphorylation and abundance in cells depleted of hSMG-1 or ATM, levels of the p53 target p21 were still elevated and the G(1) checkpoint remained intact. Conditional overexpression of p21 in p53-deficient cells revealed that hyperoxia also stimulates wortmannin-sensitive degradation of p21. siRNA depletion of hSMG-1 or ATM restored p21 stability and the G(1) checkpoint during hyperoxia. These findings establish hSMG-1 as a proximal regulator of DNA damage signaling and reveal that the G(1) checkpoint is tightly regulated during prolonged oxidative stress by both PIKK-dependent synthesis and proteolysis of p21.

The HIV-1 reverse transcriptase mutants G190S and G190A, which confer resistance to non-nucleoside reverse transcriptase inhibitors, demonstrate reductions in RNase H activity and DNA synthesis from tRNA(Lys, 3) that correlate with reductions in replication efficiency.

We evaluated the replication efficiency of the HIV reverse transcriptase (RT) mutants K103N, G190A, and G190S, which confer resistance to the non-nucleoside RT inhibitor efavirenz, using growth competition assays in cell culture. In the absence of efavirenz, the fitness hierarchy was G190S < G190A < K103N < wild-type. The fitness reduction of G190S relative to K103N was less evident at high efavirenz concentrations, although K103N still replicated more efficiently. Efficiency of RNase H cleavage and RNA-dependent DNA synthesis from tRNA(Lys, 3) correlated with relative fitness, in biochemical studies of mutant RTs. Presteady state and steady state polymerization assays using DNA primers detected no abnormalities. This work is consistent with previous studies demonstrating that initiation of viral DNA synthesis is reduced in mutants with slowed RNase H cleavage, and suggests that both abnormalities contribute to the replication defect of these mutants. It also suggests that high concentrations of efavirenz are unlikely to favor the selection of G190S clinically.

Regulatory roles of p21 and apurinic/apyrimidinic endonuclease 1 in base excision repair.

Many types of DNA damage induce a cellular response that inhibits replication but allows repair by up-regulating the p53 pathway and inducing p21(Cip1, Waf1, Sdi1). The p21 regulatory protein can bind proliferating cell nuclear antigen (PCNA) and prohibit DNA replication. We show here that p21 also inhibits PCNA stimulation of long patch base excision repair (BER) in vitro. p21 disrupts PCNA-directed stimulation of flap endonuclease 1 (FEN1), DNA ligase I, and DNA polymerase delta. The dilemma is to understand how p21 prevents DNA replication but allows BER in vivo. Differential regulation by p21 is likely to relate to the utilization of DNA polymerase beta, which is not sensitive to p21, in the repair pathway. We have also found that apurinic/apyrimidinic endonuclease 1 (APE1) stimulates long patch BER. Furthermore, neither APE1 activity nor its ability to stimulate long patch BER is significantly affected by p21 in vitro. We propose that APE1 serves as an assembly and coordination factor for long patch BER proteins. APE1 initially cleaves the DNA and then facilitates the sequential binding and catalysis by DNA polymerase beta, DNA polymerase delta, FEN1, and DNA ligase I. This model implies that BER can be regulated differentially, based upon the assembly of relevant proteins around APE1 in the presence or absence of PCNA.

Mutation of the methylated tRNA(Lys)(3) residue A58 disrupts reverse transcription and inhibits replication of human immunodeficiency virus type 1.

Cellular tRNA(Lys)(3) serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA(Lys)(3) interacts directly with HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to initiate reverse transcription. Residue A58 of tRNA(Lys)(3), which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 1-methyladenosine 58 (M(1)A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA(Lys)(3) residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA(Lys)(3) derivative, tRNA(Lys)(3)A58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M(1)A58 is necessary for the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M(1)A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M(1)A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1.

Fusion estrogen receptor proteins: toward the development of receptor-based agonists and antagonists.

Estrogen-induced signaling mediated by estrogen receptors (ERs) is also affected by aberrant ERs that act as constitutively active or dominant negative modulators. Variant ERs can contribute to carcinogenesis and to the loss of estrogen responsiveness, rendering antiestrogen therapy ineffective. Determining target gene response during co-synthesis of different ER species is difficult, because dimers formed in the presence of more than one ER species are a heterogenous population of homo- or heterodimers. We engineered a homofusion ERalpha as a prototype single-chain receptor by genetically conjugating two ER monomers into a covalently fused single-chain protein to obtain a homogeneous population. This permits analysis of symmetrical or asymmetrical mutations that simulate variant homo- and heterodimers. Although a monomer, the homofusion receptor exhibited similar biochemical and functional properties to the dimeric ERalpha. We used activation function-2 (AF2) defective mutants as a model in either one or both receptor domains for a dominant-negative phenotype by suppressing the reporter activity induced by the WT receptor. When co-expressed with ERalpha, the fusion variant deficient in both AF2 functions suppressed the reporter activity effectively induced by ERalpha. These results show the utility of fusion receptors as models for generation of receptor-based agonists and antagonists.

The kissing hairpin sequence promotes recombination within the HIV-I 5' leader region.

The role of RNA-RNA template interactions in facilitating recombination during reverse transcription of minus strand DNA has been examined. The tested hypothesis is that template switching by reverse transcriptase is promoted at sites where homologous regions of two RNAs are brought in close proximity via stable intertemplate interactions. Frequency and distribution of template switching between homologous donor and acceptor RNAs were examined within the human immunodeficiency virus type I (HIV-I) 5'-untranslated region (UTR) containing the dimer initiation sequence (DIS). Results were compared with control nondimerizing templates from the pol region. The dimerizing UTR templates displayed a 4-fold higher transfer efficiency than the control. A striking 53% of transfers in the UTR mapped near the DIS, of which two-thirds occurred immediately 5' to this sequence. In the UTR template, deletion of the DIS hairpin disrupted template dimerization and caused a 4-fold drop in transfer efficiency. Insertion of the DIS within the pol template increased both dimerization and transfer efficiency. Transfer distributions revealed that in both sets of templates, DIS-induced dimerization increased the efficiency of transfers across the whole template, with the transfers peaking around the dimerization site. Overall, these results suggest that template dimerization facilitated by the unique geometry of the DIS-promoted kissing interactions effectively promotes recombination within the HIV-I 5'-UTR.

Identification of rad27 mutations that confer differential defects in mutation avoidance, repeat tract instability, and flap cleavage.

In eukaryotes, the nuclease activity of Rad27p (Fen1p) is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments. Genetic analysis of Saccharomyces cerevisiae also has identified a role for Rad27p in mutation avoidance. rad27Delta mutants display both a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result primarily from duplications of DNA sequences that are flanked by direct repeats. These observations suggested that Rad27p activities in DNA replication and repair could be altered by mutagenesis and specifically assayed. To test this idea, we analyzed two rad27 alleles, rad27-G67S and rad27-G240D, that were identified in a screen for mutants that displayed repeat tract instability and mutator phenotypes. In chromosome stability assays, rad27-G67S strains displayed a higher frequency of repeat tract instabilities relative to CAN1 duplication events; in contrast, the rad27-G240D strains displayed the opposite phenotype. In biochemical assays, rad27-G67Sp displayed a weak exonuclease activity but significant single- and double-flap endonuclease activities. In contrast, rad27-G240Dp displayed a significant double-flap endonuclease activity but was devoid of exonuclease activity and showed only a weak single-flap endonuclease activity. Based on these observations, we hypothesize that the rad27-G67S mutant phenotypes resulted largely from specific defects in nuclease function that are important for degrading bubble intermediates, which can lead to DNA slippage events. The rad27-G240D mutant phenotypes were more difficult to reconcile to a specific biochemical defect, suggesting a structural role for Rad27p in DNA replication and repair. Since the mutants provide the means to relate nuclease functions in vitro to genetic characteristics in vivo, they are valuable tools for further analyses of the diverse biological roles of Rad27p.

DNA ligase I and proliferating cell nuclear antigen form a functional complex.

DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.

The Y181C mutant of HIV-1 reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors alters the size distribution of RNase H cleavages.

We investigated the effects of the nonnucleoside reverse transcriptase inhibitor-resistant mutant Y181C on RNA 5'-end-directed RNase H cleavage by HIV-1 reverse transcriptase, using an RNA.DNA hybrid in which a radiolabeled RNA 5' end was recessed. Y181C produced a higher ratio of secondary (9 nucleotide long) to primary (18 nucleotide long) products than wild type. When the RNA was 3'-end-labeled, Y181C generated a long product, which results when secondary cleavage precedes the primary. When using an RNA.DNA hybrid in which the labeled RNA 5' end and DNA 3' end were flush, formation of secondary product by both enzymes was inhibited. Under these conditions, Y181C cleaved closer to the RNA 5' end than wild type. Studies with this substrate labeled at the RNA 3' end showed that Y181C is no more likely than wild type to cleave toward the RNA 3' end. Thus, Y181C RT has a strong preference to cleave in the direction of the RNA 5' end even when secondary cleavage is prevented, resulting in a disruption of the normal sequence of primary followed by secondary cleavages.

Human DNA ligase I efficiently seals nicks in nucleosomes.

The access to DNA within nucleosomes is greatly restricted for most enzymes and trans-acting factors that bind DNA. We report here that human DNA ligase I, which carries out the final step of Okazaki fragment processing and of many DNA repair pathways, can access DNA that is wrapped about the surface of a nucleosome in vitro and carry out its enzymatic function with high efficiency. In addition, we find that ligase activity is not affected by the binding of linker histone (H1) but is greatly influenced by the disposition of the core histone tail domains. These results suggest that the window of opportunity for human DNA ligase I may extend well beyond the first stages of chromatin reassembly after DNA replication or repair.

Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H.

HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5' end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5' end of the RNA to make a secondary cut creating two products 8-9 nt long. RT then binds the new 5' end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3' end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.

Mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RNase H cleavage that correlate with HIV-1 replication fitness in cell culture.

Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated a three- to fourfold slowing of both DNA 3'-end-directed and RNA 5'-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J. Virol. 73:5803-5813, 1999). In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3'-end- and RNA 5'-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.

The sequential mechanism of HIV reverse transcriptase RNase H.

Synthesis of the minus strand of viral DNA by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase is accompanied by RNase H degradation of the viral RNA genome. RNA fragments remain after synthesis and are degraded by the polymerase-independent mode of RNase H cleavage. Recently, we showed that this mode of cleavage occurs by a specific ordered mechanism in which primary cuts are first, secondary and 5-nucleotide cuts are next, and second primary cuts occur last (Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P., J., and Bambara, R., A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 11978-11983). Ultimately the RNAs are cleaved into small fragments that can dissociate from the DNA template. Because the cleavage mechanism is an ordered series of events, we determined in this study whether any earlier cut is required for a later cut. By precisely inhibiting cleavage at each site, we examined the dependence of later cuts on cleavage at that site. We found that each cut is independent of the other cuts, demonstrating that the order of this stepwise mechanism is based on the rates of each cut. A mechanism for unlinked ordered cleavage consistent with these results is presented.

Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1.

Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, our results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency.

Inhibition of flap endonuclease 1 by flap secondary structure and relevance to repeat sequence expansion.

Recent genetic evidence indicates that null mutants of the 5'-flap endonuclease (FEN1) result in an expansion of repetitive sequences. The substrate for FEN1 is a flap formed by natural 5'-end displacement of the short intermediates of lagging strand replication. FEN1 binds the 5'-end of the flap, tracks to the point of annealing at the base of the flap, and then cleaves. Here we examine mechanisms by which foldback structures within the flap could contribute to repeat expansions. Cleavage by FEN1 was reduced with increased length of the foldback. However, even the longest foldbacks were cleaved at a low rate. Substrates containing the repetitive sequence CTG also were cleaved at a reduced rate. Bubble substrates, likely intermediates in repeat expansions, were inhibitory. Neither replication protein A nor proliferating cell nuclear antigen were able to assist in the removal of secondary structure within a flap. We propose that FEN1 cleaves natural foldbacks at a reduced rate. However, although the cleavage delay is not likely to influence the overall process of chromosomal replication, specific foldbacks could inhibit cleavage sufficiently to result in duplication of the foldback sequence.

Effect of flap modifications on human FEN1 cleavage.

The flap endonuclease, FEN1, plays a critical role in DNA replication and repair. Human FEN1 exhibits both a 5' to 3' exonucleolytic and a structure-specific endonucleolytic activity. On primer-template substrates containing an unannealed 5'-tail, or flap structure, FEN1 employs a unique mechanism to cleave at the point of annealing, releasing the 5'-tail intact. FEN1 appears to track along the full length of the flap from the 5'-end to the point of cleavage. Substrates containing structural modifications to the flap have been used to explore the mechanism of tracking. To determine whether the nuclease must recognize a succession of nucleotides on the flap, chemical linkers were used to replace an interior nucleotide. The nuclease could readily traverse this site. The footprint of the nuclease at the time of cleavage does not extend beyond 25 nucleotides on the flap. Eleven-nucleotide branches attached to the flap beyond the footprinted region do not prevent cleavage. Single- or double-thymine dimers also allow cleavage. cis-Platinum adducts outside the protected region are moderately inhibitory. Platinum-modified branch structures are completely inert to cleavage. These results show that some flap modifications can prevent or inhibit tracking, but the tracking mechanism tolerates a variety of flap modifications. FEN1 has a flexible loop structure through which the flap has been proposed to thread. However, efficient cleavage of branched structures is inconsistent with threading the flap through a hole in the protein.

Mutations in the primer grip region of HIV reverse transcriptase can increase replication fidelity.

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3'-ends of primers. Point mutations were created by alanine substitution at positions 224-235. Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A. Mutant W229A exhibited high fidelity and did not produce a dG:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions. This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT. We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses.

An explanation for observed estrogen receptor binding to single-stranded estrogen-responsive element DNA.

Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.

The P236L delavirdine-resistant human immunodeficiency virus type 1 mutant is replication defective and demonstrates alterations in both RNA 5'-end- and DNA 3'-end-directed RNase H activities.

The nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) delavirdine (DLV) selects in vitro for the human immunodeficiency virus type 1 (HIV-1) RT mutation P236L, which confers high-level resistance to DLV but not other NNRTIs. Unexpectedly, P236L has developed infrequently in HIV-1 isolates obtained from patients receiving DLV; K103N is the predominant resistance mutation observed in that setting. We characterized the replication fitness of viruses derived from pNL4-3 containing P236L or K103N in both H9 and primary human peripheral blood mononuclear cell cultures infected in parallel with the two mutants. In the absence of DLV, p24 production by wild-type virus occurred more rapidly and to higher levels than with either mutant; P236L consistently demonstrated a two- to threefold decrease in p24 relative to K103N. At low levels of DLV, growth of wild-type virus was severely inhibited, and K103N replicated two- to threefold more efficiently than P236L. At high concentrations of DLV, P236L replication and K103N replication were both inhibited. Recombinant RTs containing K103N or P236L were analyzed for DNA polymerization on heteropolymeric RNA templates and RNase H degradation of RNA-DNA hybrids. Neither mutant demonstrated defects in polymerization. K103N demonstrated normal RNA 5'-end-directed RNase H cleavage and slowed DNA 3'-end-directed RNase H cleavage compared to wild-type RT. P236L demonstrated slowing of both DNA 3'-end- and RNA 5'-end-directed RNase H cleavage, consistent with its reduced replication efficiency relative to K103N. These data suggest that NNRTI resistance mutations can lead to reductions in the efficiency of RNase H cleavage, which may contribute to a reduction in the replication fitness of HIV-1.

Cleavage of substrates with mismatched nucleotides by Flap endonuclease-1. Implications for mammalian Okazaki fragment processing.

Flap endonuclease-1 (FEN1) is proposed to participate in removal of the initiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the 5'-end region of the Okazaki fragment. Cleavage would occur beyond the RNA, a short distance into the DNA. The initiator RNA and an adjacent short region of DNA are synthesized by DNA polymerase alpha/primase. Because the fidelity of DNA polymerase alpha is lower than that of the DNA polymerases that complete DNA extension, mismatches occur relatively frequently near the 5'-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleotides from the 5'-end of an annealed DNA strand change the pattern of FEN1 cleavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propose that a mismatch destabilizes the helical structure over a nearby area. This allows FEN1 to cleave more efficiently, facilitating removal of the mismatch. If mismatches were not introduced during synthesis of the Okazaki fragment, helical disruption would not occur, nor would unnecessary degradation of the 5'-end of the fragment.