Methylation in eucaryotes influences the repair of G/T and A/C DNA basepair mismatches

Although methylation of DNA at some sites regulates gene expression, 5mC at many sites does not appear to have any effect. We present evidence that hemimethylation at many different sites can act as a discrimination signal in mismatch repair. Deamination of 5mC in a symmetrically methylated doublet CpG yields the mismatched base pair T/G in a hemi-methylated doublet pair. Because both bases in the mismatched pair are normal constituents of DNA, identifying the incorrect base is problematic. The only apparent distinction of the two is the methylation on the strand opposite the deamination event. Using available methylases we have produced hemi-methylated SV40 DNAs that are mismatched at a single T/G or A/C basepair in a sequence that mimics the lesion resulting from the deamination of a 5mCpG. Methylation at the adjacent cytosine results in the replacement of the T much more frequently than when no methylation is present in the heteroduplex. Cytosine methylation at sites farther removed from the mismatch is equally effective in replacing the incorrect T at the mismatch. Although methylation in vertebrates is almost exclusively on cytosine in the doublet CpG, methylation of cytosines in other doublets, as well as methylation of adenosine, also act as strand discrimination signals. Perhaps some of the excess methylation in vertebrate DNAs may serve to direct mismatch repair.     

G/U lesions are efficiently corrected to G/C in SV40 DNA

Cytosine spontaneously deaminates to form uracil, generating G/U pairs in DNA. We studied the repair of these lesions by introducing specific G/U pairs into the genome of SV40 and determining the fate of the mispaired bases in Simian cells. Analysis of 135 plaques obtained after transfection of the modified viral DNA indicates that G/U lesions were repaired to G/C in every case. This result indicates that G/U lesions are corrected with greater efficiency and specificity than any combination of DNA base/base mispairs, in transfected SV40 DNA.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

Efficient repair of A/C mismatches in mouse cells deficient in long-patch mismatch repair

A previously unrecognized mismatch repair activity is described. Extracts of immortalized MSH2-deficient mouse fibroblasts did not correct most single base mispairs. The same extracts carried out efficient repair of A/C mismatches. A/G mispairs were less efficiently corrected and there was no significant repair of A/A. MLH1-defective mouse extracts also repaired an A/C mispair. A/C correction by Msh2(-/-) mouse cell extracts was not affected by antibodies against the PMS2 protein, which inhibited long-patch mismatch repair. A/C repair activity is thus independent of MutSalpha, MutSbeta and MutLalpha. A/C mismatches were corrected 5-fold more efficiently by extracts of Msh2 knockout mouse cells than by comparable extracts prepared from hMSH2- or hMLH1-deficient human cells. MSH2-independent A/C correction by mouse cell extracts did not require a nick in the circular duplex DNA substrate. Repair involved replacement of the A and was associated with the resynthesis of a limited stretch of 相似文献   

Multiplicity of strand incision at G:T base mismatches in DNA by human cell extracts

Cell extract from the HT29 human colon carcinoma cell line (lacking mutator phenotype) was used to study the ATP-dependent G:T mismatch repair. We found that when a 45-bp (model) DNA with a single CpG/TpG mispair was incubated with the cell extract and ATP, it was incised immediately 5' and 3' to the mismatched T, and we noted that the actual 5'- and 3'-labeled fragments were similar to the cleaved products of thymine DNA glycosylase (TDG). This TDG-like cleavage product was enhanced (5-fold) with stimulation of several novel fragments, as inferred from the effect on incision at CpG/TpG site of the addition of G:U competitor DNA and ATP to the HT29 extract. The novel fragments were compatible with a strand incision on both sides of the mismatch (the third phosphodiester bond 5' and the second phosphodiester bond 3' to the mismatched T) and an incision 3' to the mismatched T, respectively. This suggests that while the ATP-dependent (TDG-like) incision activity, contrary to expectation, shows a lack of substrate competition, its catalytic property is likely modified by an interaction with G:U mispair. These multiple ATP-dependent incision events were not detected when extracts of the mismatch repair (MMR) defective HCT15 or HCT116 cell line were augmented with ATP and G:U. We postulate that these multiple ATP-dependent incision events possibly require the same MMR factors, and together they constitute a modified single ATP-dependent G:T incision activity. This activity toward the CpG/TpG was competitively inhibited by a 45-bp DNA with an ApG/TpT mispair; incision at a single site 5' to the latter mismatch compares with one of the multiple sites incised 5' to the former mismatch. These results suggest that one of several mismatch-incision factors is required by the human ATP-dependent G:T incision activity, in addition to MMR factors and ATP.     

Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches

Thermodynamic measurements are reported for 51 DNA duplexes with A.A, C.C, G.G, and T.T single mismatches in all possible Watson-Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson-Crick pair. The observed trend in stabilities of mismatches at 37 degrees C is G.G > T.T approximately A.A > C.C. The observed stability trend for the closing Watson-Crick pair on the 5' side of the mismatch is G.C >/= C.G >/= A.T >/= T.A. The mismatch contribution to duplex stability ranges from -2.22 kcal/mol for GGC.GGC to +2.66 kcal/mol for ACT.ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of DeltaG degrees 37 = 3.3%, DeltaH degrees = 7. 4%, DeltaS degrees = 8.1%, and TM = 1.1 degrees C. The imino proton region of 1-D NMR spectra shows that G.G and T.T mismatches form hydrogen-bonded structures that vary depending on the Watson-Crick context. The data reported here combined with our previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches. The results are useful for primer design and understanding the mechanism of triplet repeat diseases.     

Generation of telomeric G strand overhangs involves both G and C strand cleavage

Processing of telomeric DNA is required to generate the 3' G strand overhangs necessary for capping chromosome ends. We have investigated the steps involved in telomere processing by examining G overhang structure in Tetrahymena cells that lack telomerase or have altered telomeric sequences. We show that overhangs are generated by two precise cleavage steps involving nucleases that are robust but lack sequence specificity. Our data suggest that a G overhang binding protein delineates the boundaries for G and C strand cleavage. We also show that telomerase is not the nuclease responsible for G strand cleavage, although telomerase depletion alters the precision of processing. This change in processing indicates that telomerase affects multiple transactions at the telomere and provides a physical footprint for the continued association of telomerase with the telomere after repeat addition is complete.     

Initiation of strand incision at G:T and O(6)-methylguanine:T base mismatches in DNA by human cell extracts

Extracts of the human glioma cell line A1235 (lacking O⁶-methylguanine-DNA methyltransferase) are known to restore a G:T mismatch to a normal G:C pair in a G:T-containing model (45 bp) DNA substrate. Herein we demonstrate that substitution of G:T with O⁶-methylguanine:T (m6G:T) results in extract-induced intra-strand incision in the DNA at an efficiency comparable to that of complete repair of the G:T-containing substrate, although the m6G:T mispair serves as a poor substrate for later repair steps (e.g. gap filling, as judged by defective DNA repair synthesis). The A1235 extract, when supplemented with ATP and the four normal dNTPs, incises 5′ to the mismatched T, as inferred by the generation of a single-stranded 20mer fragment. Unlike its parental (A1235) counterpart, an extract of the alkylation-tolerant derivative cell line A1235-MR4 produces no 20mer fragment, even when thymine-DNA glycosylase (TDG) is added to the reaction mixture. In contrast, the A1235 extract, when augmented with TDG, catalyzes enhanced incision at m6G:T in the 45 bp DNA, yielding 5–10-fold greater 20mer than that of either extract or TDG alone. Interestingly, the absence of m6G:T incision activity in the A1235-MR4 extract is similar to that seen for extracts of several known mismatch repair-deficient cell lines of colon tumor origin. Together these results suggest that derivative A1235-MR4 cells are defective in m6G:T incision activity and that the efficiency of this activity in the parental (A1235) cells may depend on the presence of several ill-defined mismatch repair recognition proteins along with TDG and ATP.     

In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones

SV40-transformed cells of C57BL/6 (B6) mouse origin (H-2b) express four distinct predominant antigenic sites, I, II, III, and IV, on SV40 large tumor (T) Ag that are recognized by SV40 T Ag-specific CTL clones. In this study, we selected SV40 T Ag-positive cell lines which had lost one or more of the antigenic sites, by in vitro cocultivation of a SV40-transformed B6 mouse kidney cell line (K-0) with SV40 T Ag site-specific CTL clones, Y-1 (site I specific), Y-2 (site II specific), Y-3 (site III specific), and Y-4 (site IV specific). All of the CTL-resistant cell lines expressed large quantities of cell surface H-2 class I Ag. K-1 cells selected by CTL clone Y-1 lost the expression of antigenic sites I, II, and III, but not site IV. K-2 and K-3 cells selected by CTL clones Y-2 and Y-3, respectively, were found to be negative for sites II and III but expressed sites I and IV. K-4 cells selected by CTL clone Y-4 lost the expression of only site IV. K-1,4 cells (sites I-, II-, III-, IV-) were selected from K-1 cells by cocultivation with CTL clone Y-4, K-2,4 cells (sites I+, II-, III-, IV-) were selected from K-2 cells by CTL clone Y-4. K-3,1 cells (sites I-, II-, III-, IV+) were selected from K-3 cells by CTL clone Y-1, and K-3,1,4 cells (sites I-, II-, III-, IV-) were selected from K-3,1 cells by CTL clone Y-4. From K-4 cells, K-4,1 cells (sites I-, II-, III-, IV-) and K-4,3 cells (sites I+, II-, III-, IV-) were selected by CTL clone Y-1 and Y-3, respectively. The antigenic site loss variant cell lines K-1, K-1,4, K-3,1 K-3,1,4, K-4,1, and K-4,3 synthesized SV40 T Ag molecules of 75, 75, 78, 78, 81, and 88 kDa, respectively. Expression of wild-type SV40 T Ag in the antigenic site loss variants by infection with SV40 or transfection with cloned SV40 DNA restored the CTL recognition sites on the variant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

SV40 T antigen acts as a minor histocompatibility antigen of SV40 T antigen tolerant transgenic mice

The ability of normal mice to mount an SV40 T antigen-specific cytolytic T lymphocytes response when immunized in vivo with splenocytes from the SV40 T antigen transgenic 427-line mice and restimulated in vitro with SV40-transformed fibroblasts, or when immunized with SV40 and restimulated with 427-line splenocytes, was analyzed. Both immunization schemes resulted in an SV40 T antigen-specific immune response, indicating the presence of SV40 T antigen-positive cells in the spleens of these transgenic mice. Normal mice engrafted with skin from 427 donors showed no rejection of the graft. Thus, SV40 T antigen in transgenic 427-line mice is expressed on an undefined cell type in the spleen and acts as a tissue-specific minor histocompatibility antigen.     

Further studies of the induction and intracellular repair of DNA strand breaks using intranuclear SV40 as a test system

We have previously published the techniques and preliminary results of an SV40 viral probe assay for gamma-radiation-induced single- and double-strand DNA breaks and their intracellular repair in higher cells (Radiat. Res. 101, 356-372, 1985). Those experiments with SV40 infected CV-1 monkey kidney cells suggested that this assay technique demonstrates slow but extensive intracellular repair of single-strand breaks (SSB), and possible early repair of double-strand breaks (DSB), followed by later induction of DSB. Following up on these early observations, many additional infection-incubation experiments have now been performed with both human and simian cells. Analysis of data from these experiments involving up to 6 h of postinfection intranuclear incubation shows the same distribution of strand break damage in incubated and unincubated samples. This implies that under these experimental conditions there is neither intracellular repair nor further production of SSB or DSB in intranuclear viral DNA. We have evidence which suggests that this lack of repair or degradation occurs because the bulk of intranuclear SV40 DNA is relatively inaccessible to host cell enzymes.     

c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication.

Replicating activity of SV40 origin-containing plasmid was tested in human cells as well as in monkey CosI cells. All the plasmids possessing SV40 ori sequences could replicate, even in the absence of SV40 T antigen, in human HL-60 and Raji cells which are expressing c-myc gene at high level. The copy numbers of the replicated plasmids in these human cells were 1/100 as high as in monkey CosI cells which express SV40 T antigen constitutively. Exactly the same plasmids as the transfected original ones were recovered from the Hirt supernatant of the transfected HL-60 cells. Furthermore, replication of the SV40 ori-containing plasmids in HL-60 cells was inhibited by anti-c-myc antibody co-transfected into the cells. These results indicate that the c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication.     

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.     

A plasma membrane-associated phospholipase in SV40-transformed 3T3 cells

Cells of a SV3T3 line can be adapted to degrade phosphatides made into a film. A phosphatide degrading enzyme(s) was localized at cell surface structures by cytochemistry. Biochemistry of cell fractions indicated the presence of a phospholipase which was bound to the plasma membrane. It has a substrate specificity for phosphatidylserine, a pH optimum in the acid region, and is inhibited by calcium. It is not dissociated from the membrane by products of phospholipid degradation and is presumably a phospholipase A. The hydrolysis of the substrate under experimental conditions is about 32%. The cell biological importance of the enzyme is discussed in regard to cellular interactions and invasiveness of virus-transformed cells.     

SV40 T antigen and the exocytotic pathway.

A chimeric gene consisting of DNA coding for the 15-amino acid signal peptide of influenza virus hemagglutinin and the C-terminal 694 amino acids of SV40 large T antigen was inserted into a bovine papilloma virus (BPV) expression vector and introduced into NIH-3T3 cells. Cell lines were obtained that express high levels (approximately 5 X 10(6) molecules/cell) of the chimeric protein (HA-T antigen). The biochemical properties and intracellular localization of HA-T antigens were compared with those of wild-type T antigen. Wild-type T antigen. Wild-type T antigen is located chiefly in the cell nucleus, although a small fraction is detected on the cell surface. By contrast, HA-T antigen is found exclusively in the endoplasmic reticulum (ER). During biosynthesis, HA-T antigen is co-translationally translocated across the membrane of the ER, the signal peptide is cleaved and a mannose-rich oligosaccharide is attached to the polypeptide (T antigen contains one potential N-linked glycosylation site at Asn154). HA-T antigen does not become terminally glycosylated or acylated and little or none reaches the cell surface. These results suggest that T antigen is incapable of being transported along the exocytotic pathway. To explain the presence of wild-type T antigen on the surface of SV40-transformed cells, an alternative route is proposed involving transport of T antigen from the nucleus to the cell surface.     

The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase α-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.     

Intranuclear Redistribution of SV40T, p53, and PML in a Conditionally SV40T-Immortalized Cell Line

We have previously reported that EBNA-5, one of the Epstein–Barr virus-encoded proteins, accumulates in the nuclear bodies containing PML, the promyelocytic leukemia associated protein. In this study, we examine the intranuclear distribution of SV40 large T-antigen (SV40T), the p53 tumor suppressor protein (p53), and PML in a conditionally immortalized cell line, IDH4. In IDH4 cells, the expression of SV40T is regulated by a dexamethasone (Dex)-driven promoter. Withdrawal of Dex results in down-regulation of SV40T and growth arrest, whereas addition of Dex to the growth-arrested cells results in up-regulation of SV40T and proliferation. In proliferating IDH4 cells, SV40T is concentrated in nuclear dots that are also positive for p53. Many of these dots are juxtaposed to PML positive structures but do not colocalize with them. After removal of Dex, SV40T–p53 dots gradually disappear, while the PML structures remain. Induction of SV40T in nonproliferating IDH4 cells causes a coordinated redistribution of SV40T and p53. The immunostaining for SV40T and p53 is first weak, then strong with a homogeneous distribution, and 3–4 days later becomes dot-like again. This reappearance of SV40T–p53 dots coincides with the recovery of proliferation in restimulated IDH4 cells. Also, the p53 pattern correlates with the SV40T pattern with regard to both morphology and intensity during both suppression and induction of SV40T. Taken together, our data suggest that (i) the level of p53 is coregulated with the level of SV40T in a dose-dependent fashion; (ii) the formation of SV40T–p53 nuclear dots correlates with the transformed phenotype; (iii) the SV40T–p53 dots localize preferentially to the neighborhood of PML bodies which are already present in normal cells.