Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein.

A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells. Western blot (immunoblot) analysis using polyclonal antibodies to the E. coli MutY protein detected a protein of 65 kDa in both extracts. Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies. Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes). A nicking activity that is either associated with or copurified with MYH was also detected. Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity. The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches. No nicking activity was detected on substrates containing other selected mismatches or homoduplexes. Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters. Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates. Binding is sevenfold greater on A/GO substrates than on A/G substrates. The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E. coli MutY protein.

Differential DNA recognition and glycosylase activity of the native human MutY homolog (hMYH) and recombinant hMYH expressed in bacteria.

Human MutY homolog (hMYH), an adenine DNA glycosylase, can effectively remove misincorporated adenines opposite template G or 8-oxoG bases, thereby preventing G:C-->T:A transversions. Human cell extracts possess the adenine DNA glycosylase activity of hMYH and can form protein-DNA complexes with both A/G and A/8-oxoG mismatches. hMYH in cell extracts was shown to be the primary binding protein for A/G- and A/8-oxoG-containing DNA substrates by UV cross-linking. However, recombinant hMYH expressed in bacteria has much weaker glycosylase and substrate-binding activities towards A/G mismatches than native hMYH. Moreover, the protein-DNA complex of bacterially expressed hMYH migrates much faster than that of native hMYH in a non-denaturing polyacrylamide gel. Dephosphorylation of native hMYH reduces the glycosylase activity on A/G more extensively than on A/8-oxoG mismatches but does not alter the gel mobility of the protein-DNA complex. Our results suggest that hMYH in human cell extracts may be associated with other factors in the protein-DNA complex to account for its slower mobility in the gel. hMYH and apurinic/apyrimidinic endonuclease (hAPE1) co-migrate with the protein-DNA complex formed by the extracts and A/8-oxoG-containing DNA.

Intact MutY and its catalytic domain differentially contact with A/8-oxoG-containing DNA.

Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase active on DNA substrates containing A/G, A/8-oxoG, A/C or G/8-oxoG mismatches. A truncated form of MutY (M25, residues 1-226) retains catalytic activity; however, the C-terminal domain of MutY is required for specific binding to the 8-oxoG and is critical for mutation avoidance of oxidative damage. Using alkylation interference experiments, the determinants of the truncated and intact MutY were compared on A/8-oxoG-containing DNA. Several purines within the proximity of mismatched A/8-oxoG show differential contact by the truncated and intact MutY. Most importantly, methylation at the N7 position of the mismatched 8-oxoG and the N3 position of mismatched A interfere with intact MutY but not with M25 binding. The electrostatic contacts of MutY and M25 with the A/8-oxoG-containing DNA substrates are drastically different as shown by ethylation interference experiments. Five consecutive phosphate groups surrounding the 8-oxoG (one on the 3' side and four on the 5' side) interact with MutY but not with M25. The activities of the truncated and intact MutY are modulated differently by two minor groove-binding drugs, distamycin A and Hoechst 33258. Both distamycin A and Hoechst 33258 can inhibit, to a similar extent, the binding and glycosylase activities of MutY and M25 on A/G mismatch. However, binding and glycosylase activities on A/8-oxoG mismatch of intact MutY are inhibited to a lesser degree than those of M25. Overall, these results suggest that the C-terminal domain of MutY specifies additional contact sites on A/GO-containing DNA that are not found in MutY-A/G and M25-A/8-oxoG interactions.

Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria.

A protein homologous to the Escherichia coli MutY glycosylase, referred to as mtMYH, has been purified from calf liver mitochondria. SDS-polyacrylamide gel electrophoresis, western blot analysis as well as gel filtration chromatography predicted the molecular mass of the purified calf mtMYH to be 35-40 kDa. Gel mobility shift analysis showed that the purified mtMYH formed specific binding complexes with A/8-oxoG, G/8-oxoG and T/8-oxoG, weakly with C/8-oxoG, but not with A/G and A/C mismatches. The purified mtMYH exhibited DNA glycosylase activity removing adenine mispaired with G, C or 8-oxoG and weakly removing guanine mispaired with 8-oxoG. The mtMYH glycosylase activity was insensitive to high concentrations of NaCl and EDTA. The purified mtMYH cross-reacted with antibodies against both intact MutY and a peptide of human MutY homolog (hMYH). DNA glycosylase activity of mtMYH was inhibited by anti-MutY antibodies but not by anti-hMYH peptide antibodies. Together with the previously described mitochondrial MutT homolog (MTH1) and 8-oxoG glycosylase (OGG1, a functional MutM homolog), mtMYH can protect mitochondrial DNA from the mutagenic effects of 8-oxoG.

Repair of single base-pair transversion mismatches of Escherichia coli in vitro: correction of certain A/G mismatches is independent of dam methylation and host mutHLS gene functions.

Six different base-pair transversion mismatches are repaired with different efficiencies in an in vitro mismatch repair system. In particular, the T/T and C/C mismatches appear to be less efficiently repaired than the A/A and G/G mismatches. Four A/G and four C/T mismatches at different positions are repaired to different extents. One of the A/G mismatches is repaired equally efficiently when DNA heteroduplexes are fully methylated or hemi-methylated at the d(GATC) sequences. This type of mismatch repair appears to be unidirectional with A to C conversion by acting at A/G mispairs to restore the C/G pairs. This methylation-independent correction is not controlled by the mutH, mutL, mutS, uvrE, uvrB, phr, recA, recF, and recJ gene products. The independence of the transversion mismatch repair of these genes and methylation distinguishes this from the known mismatch repair pathways.

Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus.

A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.

Repair of oxidative DNA damage: mechanisms and functions.

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2-6-nucleotide patch size) pathways need AP endonuclease to generate a 3' hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.

The dimer-tetramer equilibrium of recombinant hemoglobins. Stabilization of the alpha 1 beta 2 interface by the mutation beta(Cys112-->Gly) at the alpha 1 beta 1 interface.

The dimer-tetramer association constants of several recombinant human hemoglobins (in the CO form) have been measured by differential gel filtration. Recombinant human hemoglobin prepared from recombinant beta-chains, and mutant hemoglobins where the substitution was on the surface, beta(Thr4-->Asp), in the heme pocket, beta(Val67-->Thr), at the 2,3-DPG binding site, beta(Val1-->Met+His2del), had a twofold smaller association with respect to natural hemoglobin. In a mutant at the alpha 1 beta 2 interface, beta(Cys93-->Ala), the association constant was decreased three-fold. Conversely, in a mutant at the alpha 1 beta 1 interface, beta(Cys112-->Gly), the association constant was two- and four-fold increased with respect to natural and recombinant human hemoglobin. These differences are energetically very small, consistent with the correct folding of the recombinant hemoglobins. The stabilization of the tetrameric structure by a mutation at the alpha 1 beta 1 interface indicates that structural changes in this interface can be propagated through the protein to the alpha 1 beta 2 interface and, thereby, exert an effect on the allosteric equilibrium.

[Changes in the content and activity of HSC70/HSP68, proteinases and phosphatases during liver regeneration].

This article reports changes in acid and alkaline phosphatases (ACP and AKP), constitutive heat shock protein 70/induced heat shock protein 68 (HSC70/HSP68) and acid and neutral proteinases during liver regeneration (0-144 h) after 2/3 hepatectomy (partial hepatectomy, PH). Both ACP and AKP had two active peaks at 4 and 48 h, 16 and 96 h, respectively, which were followed by significant decrease. The content of HSC70/HSP68 also showed two peaks (16 and 96 h), of which the content after the second peak decreased more obviously than after the first peak. Moreover a 90 kD neutral proteinase was induced at the time from 2 to 6 h and a 27 kD acid proteinase was induced at 36 h. The results suggest that the ACP and the 90 kD neutral proteinase may participate in activating hepatocytes from G0-phase into G1-phase, and AKP and HSC70/HSP68 may play a role mainly in DNA synthesis, cellular metabolism and proliferation. Furthermore 27 kD acid proteinase may be involved in re-differentiation of hepatocytes and reconstruction of liver tissue.

Tembusu virus infection in Cherry Valley ducks: the effect of age at infection.

Three groups of Cherry Valley ducks at 5 day, 2 week and 5 week of age were intranasally infected with the WFCL strain of Tembusu virus (TMUV) to investigate the effect of host age on the outcome of TMUV infection. For each age group, clinical signs, gross and microscopic lesions, viral copy numbers in tissues and serum neutralizing antibody titers were recorded. Age-related differences in the resistance to TMUV infection were observed with younger ducks being more susceptible. Some ducks infected at 5 day and 2 week of age developed severe clinical signs, including severe neurological dysfunction and death. However, subclinical signs and no mortality were observed in ducks infected at 5 week of age. A decline in the severity of gross and microscopic lesions was observed as ducks mature. Systemic infections were established in the three age groups post challenge. Higher viral copy numbers in the tissues, especially in vital organs such as the brain and the heart, were developed in the ducks infected at 5 day of age than older ducks, correlating with the severity of clinical signs and lesions in the tissues. Furthermore, ducks infected at 5 week of age developed significantly higher serum neutralizing antibody titers than ducks infected at 5 day of age as determined by serum neutralization test. Therefore, age-related differences in the resistance to TMUV infection should be considered when studying the pathogenicity, pathogenesis, formulation of the vaccination and therapy strategies of TMUV infection in ducks.

Kashin-Beck disease and Sayiwak disease in China: prevalence and a comparison of the clinical manifestations, familial aggregation, and heritability.

OBJECTIVE: To compare the prevalence, the clinical manifestations, familial aggregation and heritability of Kashin-Beck disease (KBD) and Sayiwak disease (SD) in China. METHODS: 10,823 people from 1361 families in 14 villages in Linyou County, Shaanxi Province, were examined for KBD, and 2264 people from 552 families in Sayiwak village, Kashi city, Xinjiang, were examined for SD. The investigation included documentation of individual information and clinical manifestations. Patients were subject to radiographic imaging of the right hand. t-Tests and chi-square tests were used to examine correlations of the diseases with age and gender in each of the two groups. Analysis of familial aggregation was conducted with the chi-square distribution analysis of goodness of fit using the SAS8.0 program. The Li-Mantel-Gart method was employed for the segregation analysis. The Falconer regression method11 was employed to estimate heritability (h²). RESULTS: The prevalence of KBD in Linyou County was 10.90%, and of SD in Sayiwak village was 0.57%. Of the 21 clinical signs examined, KBD cases exhibited 19 signs (90.48%) and SD cases exhibited 18 signs (85.71%), which indicate similarities between the two diseases. However, different clinical signs were evident between the KBD and SD cases, with different impairment rates among joints of limbs in KBD and similar rates in SD. A comparison of radiological features of limb arthropathy between the two diseases showed differences in several characteristics between the two diseases. In addition, measurements of stature and sitting height showed significant differences in bone development between the two diseases. For KBD cases, the values of h² in the first-degree and the second-degree relatives were 41.76% and 37.20% (P<0.05). The CI of h² was 31.17-52.38 and 19.86-54.55, with a segregation ratio of P=0.12, SE(P)=0.014, 95%CI 0.09-0.15, less than 0.25(χ²=42.36, df=1, P<0.001). For SD cases, the values of h² were 155.61%, 273.63% and 236.83%. The 95% CIs of h² were 133.20-178.12, 229.83-317.42 and 145.83-327.81, respectively, with a segregation ratio of P=0.34, SE(P)=0.059, and CIs between 0.22 and 0.45(χ²=4.9817, df=1, P>0.05). CONCLUSION: The results indicate both similarities and differences in the clinical manifestations of KBD and SD. However, environmental factors appear to play a major role in KBD, while hereditability is a major factor in SD.

Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes.

Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.

Influence of GATC sequences on Escherichia coli DNA mismatch repair in vitro.

The effect of the number and position of DNA adenine methylation (dam) sites, i.e., d(GATC) sequences, on mismatch repair in Escherichia coli was investigated. The efficiency of repair was measured in an in vitro assay which used an f1 heteroduplex containing a G/T mismatch within the single EcoRI site. Both an increase in the number of dam sites and a shortened distance between dam site and mismatched site increased the efficiency of mismatch repair. The sequences adjacent to d(GATC) also affected the efficiency of methylation-directed mismatch repair. Furthermore, heteroduplexes with one extra dam site located close to either the 5' or 3' end of the excised base increased the repair efficiency to about the same extent. The findings suggest that the mismatch repair pathway has no preferred polarity.

A novel nucleotide excision repair for the conversion of an A/G mismatch to C/G base pair in E. coli.

A protein that binds specifically to A/G mismatches has been detected in E. coli by a gel electrophoresis DNA binding assay. A specific endonuclease is associated with the A/G mismatch-binding protein through two chromatographic steps. The endonuclease is specific for A/G-containing DNA fragments and has no cleavage activity on DNA containing the other seven possible mispairs or homoduplex DNA. The endonuclease simultaneously makes incisions at the first phosphodiester bond 3' to and the second phosphodiester bond 5' to the dA of the A/G mismatch. No incision site was detected on the other strand. These results are consistent with the unidirectional A to C conversion and short repair tract of a novel dam- and mutHLS-independent A/G repair pathway we have recently described. A nucleotide excision repair model is proposed for the conversion of an A/G mismatch to a C/G base pair.

Base mismatch-specific endonuclease activity in extracts from Saccharomyces cerevisiae.

An endonuclease activity (called MS-nicking) for all possible base mismatches has been detected in the extracts of yeast, Saccharomyces cerevisiae. DNAs with twelve possible base mismatches at one defined position are cleaved at different efficiencies. DNA fragments with A/G, G/A, T/G, G/T, G/G, or A/A mismatches are nicked with greater efficiencies than C/T, T/C, C/A, and C/C. DNA with an A/C or T/T mismatch is nicked with an intermediate efficiency. The MS-nicking is only on one particular DNA strand, and this strand disparity is not controlled by methylation, strand break, or nature of the mismatch. The nicks have been mapped at 2-3 places at second, third, and fourth phosphodiester bonds 5' to the mispaired base; from the time course study, the fourth phosphodiester bond probably is the primary incision site. This activity may be involved in mismatch repair during genetic recombination.

Detection of single DNA base mutations with mismatch repair enzymes.

A novel method for identifying DNA point mutations has been developed by using mismatch repair enzymes. The high specificity of the Escherichia coli MutY protein has permitted the development of a reliable and sensitive method for the detection and characterization of point mutations in the human genome. The MutY protein is involved in a repair pathway that can convert A/G or A/C mismatches to C/G or G/C basepairs, respectively. A/G or A/C mismatches formed by hybridization between two amplified genomic DNA samples or between specific DNA probes and target DNA are nicked at the mispaired adenine strand by MutY protein. As little as 1% of the mutant sequence can be detected by the mismatch repair enzyme cleavage (MREC) method in a mixture of normal and mutated DNAs (e.g., mutant cells are only present in 1% of the normal cell background). By using different probes, the assay also can determine the nucleotide sequence of the mutation. We have applied this method to detect single-base substitutions in human oncogenes.

Molecular cloning and functional analysis of the MutY homolog of Deinococcus radiodurans.

The mutY homolog gene (mutY(Dr)) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutY(Ec)) protein. Expressed MutY(Dr) is able to complement E. coli mutY mutants but not mutM mutants to reduce the mutation frequency. The glycosylase and binding activities of MutY(Dr) with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutY(Ec) protein, purified recombinant MutY(Dr) expressed in E. coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutY(Dr) exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutY(Dr) at the position corresponding to the position of MutY(Ec) Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutY(Dr) are similar to those of MutY(Ec). Although MutY(Dr) has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutY(Ec) does, the binding affinities for both mismatches are slightly lower for MutY(Dr) than for MutY(Ec). Thus, MutY(Dr) can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.

Characterization of the recombinant MutY homolog, an adenine DNA glycosylase, from yeast Schizosaccharomyces pombe.

The mutY homolog (SpMYH) gene from a cDNA library of Schizosaccharomyces pombe encodes a protein of 461 amino acids that displays 28 and 31% identity to Escherichia coli MutY and human MutY homolog (MYH), respectively. Expressed SpMYH is able to complement an E. coli mutY mutant to reduce the mutation rate. Similar to E. coli MutY protein, purified recombinant SpMYH expressed in E. coli has adenine DNA glycosylase and apurinic/apyrimidinic lyase activities on A/G- and A/7,8-dihydro-8-oxoguanine (8-oxoG)-containing DNA. However, both enzymes have different salt requirements and slightly different substrate specificities. SpMYH has greater glycosylase activity on 2-aminopurine/G and A/2-aminopurine but weaker activity on A/C than E. coli MutY. Both enzymes also have different substrate binding affinity and catalytic parameters. Although SpMYH has great affinity to A/8-oxoG-containing DNA as MutY, the binding affinity to A/G-containing DNA is substantially lower for SpMYH than MutY. SpMYH has similar reactivity to both A/G- and A/8-oxoG-containing DNA; however, MutY cleaves A/G-containing DNA about 3-fold more efficiently than it does A/8-oxoG-containing DNA. Thus, SpMYH is the functional eukaryotic MutY homolog responsible for reduction of 8-oxoG mutational effect.

Nucleotide sequence of a 5S ribosomal RNA gene in the sea urchin Lytechinus variegatus.

The nucleotide sequence of a cloned DNA fragment corresponding to a gene for 5S ribosomal RNA from the sea urchin Lytechinus variegatus has been determined. This sequence is representative of the dominant species of 5S rRNA labelled in vivo with 32pO4 during the cleavage stage of Lytechninus embryonic development.