Genetic Evidence for Physical Interactions between Enzymes of Nucleotide Synthesis and Proteins Involved in DNA Replication in Bacteriophage T4

We have found that mutations in phage T4 genes 41 (five of five) and 61 (three of three) cause resistance to the folate analogue pyrimethamine that inhibits T4 dihydrofolate (FH₂) reductase. These genes code for subunits of a T4 primase and are part of a putative T4 replication complex. In contrast to many previously isolated folate analogue-resistant (Far) T4 mutants, these T4 primase mutants do not overproduce FH₂ reductase nor do they alter its primary structure. A new mutant with a single lesion in gene 41 was isolated which proved resistant to the folate analogue at 30° and was lethal at 42°. This mutant induced normal levels of FH₂ reductase (encoded by the frd gene) and appeared to have normal expression of other T4 genes at 30°. Like other mutations in gene 41, tsP129 reduced phage-induced DNA synthesis to about 15% that of wild-type T4 as measured by thymidine incorporation under restrictive conditions. Double mutants carrying mutations in genes 41 and 61, 41 and frd or 61 and frd showed allele-specific suppression suggesting that the products of these genes interact. We suggest that abnormal interactions between components of the replication complex and a DNA precursor synthesizing complex cause folate analog resistance by allosterically altering the T4 FH₂ reductase.     

Hyperproduction of dihydrofolate reductase in Diplococcus pneumoniae by mutation in the structural gene: The absence of an effect on other enzymes of folate coenzyme biosynthesis

A unique group of mutations (ame^r) in the dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP⁺ oxidoreductase, EC 1.5.1.3.) structural gene of Diplococcus pneumoniae determine a marked overproduction of the corresponding enzyme protein. Since findings with these mutations relate to a key metabolic function and may be important to the regulation of folate coenzyme synthesis in general, the same group of multations were also examined for their effects on a number of related enzymic activities. Mutant and wild-type cell-free extracts, in addition to dihydrofolate reductase activity, exhibited both dihydropteroate and dihydrofolate synthetic activities under the conditions employed. Four folate coenzyme-related enzyme activities could also be demonstrated with the same preparations. These are mediated by the following enzymes, serine hydroxymethyl transferase (l-serine: tetrahydrofolate 10-hydroxymethyl tranferase, EC 2.1.2.1), 5, 10-methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.5), 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) and glutamate formiminotransferase (N-formimino-l-glutamate: tetrahydrofolate 5-formiminotransferase, EC 2.1.2.5). The ame^r mutations examined in the current study determined 3–80-fold increases in dihydrofolate reductase in comparison to the wild type. However, none of the other folate-related enzyme activities were altered. The possible significance of these findings in light of previous results is discussed.     

Regulation of the synthesis of bacteriophage T4 gene 32 protein

The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment.Mutations in genes required for late gene expression (genes 33 and 53) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not dependent on DNA synthesis.These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by its own function. Thus, in the absence of P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently in the maturation of replicated DNA, the increased P32 production is caused by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41, 56, 58, 60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.     

Properties of Bacteriophage T4 Mutants Defective in Gene 30 (Deoxyribonucleic Acid Ligase) and the rII Gene

In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.     

Studies on the Recombination Genes of Bacteriophage T4: Suppression of uvsX and uvsY Mutations by uvsW Mutations

Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42 degrees and were pseudorevertants containing a new mutation in uvsW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41 degrees the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30 degrees it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41 degrees was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product.     

Protein-DNA interactions in the T4 dNTP synthetase complex dependent on gene 32 single-stranded DNA-binding protein

Our laboratory has reported data suggesting a role for T4 phage gene 32 single-stranded DNA-binding protein in organizing a complex of deoxyribonucleotide-synthesizing enzymes at the replication fork. In this article we examined the effects of gene 32 ablation on the association of these enzymes with DNA-protein complexes. These experiments showed several deoxyribonucleotide-synthesizing enzymes to be present in DNA-protein complexes, with some of these associations being dependent on gene 32 protein. To further understand the role of gp32, we created amber mutations at codons 24 and 204 of gene 32, which encodes a 301-residue protein. We used the newly created mutants along with several experimental approaches--DNA-cellulose chromatography, immunoprecipitation, optical biosensor analysis and glutathione-S-transferase pulldowns--to identify relevant protein-protein and protein-DNA interactions. These experiments identified several proteins whose interactions with DNA depend on the presence of intact gp32, notably thymidylate synthase, dihydrofolate (DHF) reductase, ribonucleotide reductase (RNR) and Escherichia coli nucleoside diphosphate (NDP) kinase, and they also demonstrated direct associations between gp32 and RNR and NDP kinase, but not dCMP hydroxymethylase, deoxyribonucleoside monophosphate kinase, or DHF reductase. Taken together, the results support the hypothesis that the gene 32 protein helps to recruit enzymes of deoxyribonucleoside triphosphates synthesis to DNA replication sites.     

亚甲基四氢叶酸还原酶(C677/T)基因的单核苷酸多态性

亚甲基四氢叶酸还原酶(methylene tetrahydrofolatucte redase,MTHFR)是叶酸代谢过程中的关键酶,对叶酸和同型半胱氨酸的代谢以及DNA的合成、修复与甲基化均有重要作用。MTHFR基因变异导致酶热稳定性及活性降低,引起相关代谢及DNA甲基化异常,进而发生相关疾病。MTHFR具有多种变异型,本文对其中常见的一种C677T的多态性及其与疾病的相关性的研究进展做一综述。     

Reversion of Frameshift Mutations Stimulated by Lesions in Early Function Genes of Bacteriophage T4

Temperature-sensitive (ts) mutants representative of a number of genes of phage T4 were crossed with rII mutants to allow isolation of ts, rII double-mutant recombinants. The rII mutations used were characterized as frameshift mutations primarily on the basis of their revertability by proflavine. For each ts, rII double mutant, the effect of the ts mutation on spontaneous reversion of the rII mutation was determined over a range of incubation temperatures. A strong enhancement in reversion of two different rII mutants was detected when they were combined with tsL56, a mutation in gene 43 [deoxyribonucleic acid (DNA) polymerase]. Three other mutants defective in gene 43 enhanced reversion about fourfold. Two mutations in gene 32, which specifies a protein necessary for DNA replication, enhanced reversion about 5-fold and 18-fold, respectively. Two additional mutations in gene 43 and two in gene 32 had no effect. Fivefold and threefold enhancements in reversion were also found with mutations in genes 44 (DNA synthesis) and 47 (deoxyribonuclease), respectively. No significant effect was found with mutations in seven additional genes. The results of other workers suggest that frameshift mutations arise from errors in strand alignment during repair synthesis occurring at chromosome tips. Our results show that such errors can be enhanced by mutations in the DNA polymerase, the gene 32 protein, and the enzymes specified by genes 44 and 47. This implies that these proteins are employed in the repair process occurring at chromosome tips and that mutational errors in these proteins can lead to loss of ability to recognize and reject strand misalignments.     

Gene 32 protein of bacteriophage T4 moderates the activities of the T4 gene 46/47-controlled nuclease and of the Escherichia coli RecBC nuclease in vivo.

Genes 46 and 47 of phage T4 control a nuclease that is required for genetic recombination and may act similarly to the Escherichia coli RecBC nuclease. In vivo, the nucleolytic activities of both of these nucleases must be moderated so that recombining DNA intermediates are not destroyed. We conclude from our present experiments that the phage T4 gene 32 protein, specifically its C-terminal domain, participates in such moderation. We have investigated DNA degradation in different gene 32 and gene 32/46 mutants under conditions that are completely restrictive for progeny production in all the mutants. Under these conditions, DNA of those gene 32 mutants in which the C-terminal domain of the protein is not synthesized or is modified is degraded to acid-soluble material. T4 gene 46 or E. coli recB mutations reduce such degradation; together they abolish it completely. By contrast, single gene 32 mutants which produce an unaltered C-terminal domain show little or no degradation of their DNA. Residual protection against nucleases is unrelated to residual primary DNA replication or to overproduction of the mutant peptides in the different gene 32 mutants.     

Lack of dihydrofolate reductase activity in brain tissue of mammalian species: possible implications

IT IS becoming increasingly clear that folates play a vital, yet until recently an unrecognized, role in the development and function of the brain. Thus several groups of patients have been found with severe maldevelopment of the brain and mental retardation associated with inborn errors of folate metabolism resulting from congenital deficiency in one or more enzymes involved in folate metabolism (ARAKAWA et al., 1965; 1966; 1967; MUDD, LEVY and ABELES, 1969; ARAKAWA, 1970). The presence of folate coenzymes in brain tissue has been reported by several investigators (ALLEN and KLIPSTEIN, 1970; MCCLAIN and BRIDGERS, 1969). MCCLAIN and BRIDGERS (1969) showed that much less of the folates in brain are in the form of the N5-methyl derivatives than is the case for folates in plasma, red blood cells and liver. Appreciable activity of several folate interconverting enzymes have been demonstrated in brain tissue; for example, N5-methyl tetrahydrofolate homocysteine methyl transferase has been found to exist in higher levels in brain than in liver or kidney (MANGUM, 1972); N5-methyl FH,N-dimethyl-dopamine methyl transferase (LADURON, 1972) and serine transhydroxymethylase (EC 2.1.1; L-Serine: tetrahydrofolate 5, 10-hydroxymethyl transferase) (BRIDGERS, 1968) have recently been detected in brain. The last enzyme is known to catalyse a reaction responsible for the generation of a major portion of one-carbon units. In mouse brain, the activity of this enzyme declines during the first 2 weeks of extra-uterine life (BRIDGERS, 1968). The aim of the present study was to determine the levels of dihydrofolate reductase(5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) in mammalian brain tissues in comparison to the levels in other tissues. This enzyme occupies the first and key position in folate metabolism, reducing the metabolically inert vitamin, folic acid, to tetrahydrofolate. This enzyme also functions in thymidylate synthesis to regenerate tetrahydrofolate from dihydrofolate, a product of the reaction (HWHREYS and GREENBERG, 1958). In this reduced state the molecule can accept one-carbon units from various sources to give rise to metabolically active coenzyme forms of folate. This communication reports the complete absence of dihydrofolate reductase in brain tissue of several mammalian species.     

Biochemical and genetic analysis of methylenetetrahydrofolate reductase in Leishmania metabolism and virulence

Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is the sole enzyme responsible for generation of 5-methyltetrahydrofolate, which is required for methionine synthesis and provision of methyl groups via S-adenosylmethionine. Genome analysis showed that Leishmania species, unlike Trypanosoma brucei and Trypanosoma cruzi, contain genes encoding MTHFR and two distinct methionine synthases. Leishmania MTHFR differed from those in other eukaryotes by the absence of a C-terminal regulatory domain. L. major MTHFR was expressed in yeast and recombinant enzyme was produced in Escherichia coli. MTHFR was not inhibited by S-adenosylmethionine and, uniquely among folate-metabolizing enzymes, showed dual-cofactor specificity with NADH and NADPH under physiological conditions. MTHFR null mutants (mthfr(-)) lacked 5-methyltetrahydrofolate, the most abundant intracellular folate, and could not utilize exogenous homocysteine for growth. Under conditions of methionine limitation mthfr(-) mutant cells grew poorly, whereas their growth was normal in standard culture media. Neither in vitro MTHFR activity nor the growth of mthfr(-) mutants or MTHFR overexpressors were differentially affected by antifolates known to inhibit parasite growth via targets beyond dihydrofolate reductase and pteridine reductase 1. In a mouse model of infection mthfr(-) mutants showed good infectivity and virulence, indicating that sufficient methionine is available within the parasitophorous vacuole to meet the needs of the parasite.     

Isolation of Mutants Defective in α-Amylase from Bacillus subtilis: Genetic Analyses

The rate of alpha-amylase (EC 3.2.1.1) synthesis in Bacillus subtilis is regulated by a gene, amyR, located near a structural gene, amyE, for the enzyme. To construct a fine map of the amyR-amyE region, we isolated 28 mutants defective in alpha-amylase activity. Eleven mutants out of 28 showed no alpha-amylase activity, whereas the other 17 showed less alpha-amylase activity than the parent. Out of 17 partially positive alpha-amylase mutants, 10 produced temperature-sensitive enzymes, and 4 produced immunologically altered enzymes, two of which are concurrently temperature-sensitive, and 5 produced smaller amounts of alpha-amylases which are indistinguishable from normal enzyme in their temperature sensitivity and immunological properties. Two out of 11 alpha-amylase-negative mutants produced material that cross-reacted with anti-amylase serum, and 3 mutants carried suppressible mutations by the suppressor described by Okubo. Mapping data indicate that all 28 mutation sites are located in the amyE region, and none of the groups of the mutants mentioned above contains lesions that are clustered in a single region of amyE. The amyR gene seems most likely to adjoin the terminal region of amyE.     

Biochemistry of DNA-defective mutants of bacteriophage T4. VI. Biological functions of gene 42.

Bacteriophage T4 gene 1 and 42 amber mutants (defective in deoxynucleoside monophosphate kinase and deoxycytidylate hydroxymethylase, respectively) are able to synthesize DNA in cell-free lysates prepared as described by Barry and Alberts (1972), in contrast to their inabliity to do so in plasmolyzed and toluenized cell systems. Addition of extracts containing an active gene 1 or 42 product has no effect on synthesis in lysates defective in the respective gene. Thus, if these enzymes do play additional direct roles in replication, these roles are not manifest in the lysed-cell system. The gene 42 mutant am N122/m, a double mutant bearing an additional defect in DNA polymerase, is unable to synthesize DNA in these lysates. This inability is overcome by addition of extracts containing an active T4 DNA polymerase. m is a leaky amber mutation which reduces DNA polymerase activity to a very low level. However, this level is high enough to allow positive genetic complementation tests with gene 43 mutants. Two other gene 42 amber mutants contain additional defects: am 269 induces only half the normal level of DNA polymerase, and am C87 fails to induce a detectable level of thymidylate synthetase. These defects do not result from pleiotropic effects of the gene 42 mutations. In plasmolyzed cells, temperature-sensitive gene 42 mutants fail to synthesize DNA under conditions where replication forks and 5-hydroxymethyl-dCTP are present. This supports the idea that the gene 42 protein is directly involved in DNA synthesis.     

Evidence for the essential function of 2,4-dienoyl-coenzyme A reductase in the beta-oxidation of unsaturated fatty acids in vivo. Isolation and characterization of an Escherichia coli mutant with a defective 2,4-dienoyl-coenzyme A reductase

For the purpose of assessing in vivo the importance of 2,4-dienoyl-CoA reductase (EC 1.3.1.34) in the beta-oxidation of unsaturated fatty acids, reductase mutants of Escherichia coli were isolated by selecting cells that were able to grow on oleate but not on petroselinic acid (6-cis-octadecenoic acid). One mutant (fadH) exhibited 12% of the 2,4-dienoyl-CoA reductase activity present in the parental strain with other beta-oxidation enzymes being essentially unaffected. Antireductase antibodies were used to show that the mutant contains a fadH gene product at a level similar to that observed in the parental strain. Thus, the mutation seems to have resulted in the synthesis of a fadH gene product with lower specific activity. The mutation was mapped in the 71-75-min region of the E. coli chromosome where no other gene for beta-oxidation enzymes has so far been located. Complementation of the mutation by F'141, which carries the 67-75.5-min region of the E. coli genome, resulted in an increase in the 2,4-dienoyl-CoA reductase activity to 80% of the level found in the parental strain. Measurements of respiration with petroselinic acid as the substrate showed rates to be linearly dependent on the 2,4-dienoyl-CoA reductase activity up to levels found in wild-type E. coli. 2,4-Dienoyl-CoA reductase, like other enzymes of beta-oxidation, was induced when E. coli was grown on a long chain fatty acid as the sole carbon source. It is concluded that 2,4-dienoyl-CoA reductase is required in vivo for the beta-oxidation of unsaturated fatty acids with double bonds extending from even-numbered carbon atoms.     

Cloning and characterization of the Escherichia coli lit gene, which blocks bacteriophage T4 late gene expression.

Escherichia coli lit mutations inhibit gene expression late in infection by bacteriophage T4. We cloned the lit gene from wild-type E. coli and three independent lit mutants. We present evidence that lit mutations [renamed lit(Con) mutations] cause overproduction of the lit gene product and that overproduction of this product causes the inhibition of gene expression. We also present evidence that the lit gene product is nonessential for E. coli growth, although the gene is common to most E. coli K-12 strains.     

Escherichia coli mutants with temperature-sensitive synthesis of DNA

Summary Seven mutants of E. coli with temperature-sensitive synthesis of DNA have been isolated. Synthesis of RNA, protein and DNA precursors does not appear to be directly affected. The mutants can be divided into at least two groups on the basis of their pattern of DNA synthesis, their ability to support phage growth at 41° and their genetic mapping.Mutants of the first group are heterogeneous in their pattern of DNA synthesis at 40°. Some mutants cease DNA synthesis abruptly upon transfer to 40° and any residual DNA synthesis is barely detectable. In others there is substantial residual synthesis at 40°. All these Group 1 mutants are alike, however, in that they support the growth of phage T4 but not Lambda at 41°. Two mutants with barely detectable residual DNA synthesis carry DNA mutations which have been mapped by P1 transduction and show about 72% linkage to the malB locus. It has not yet proved possible to map accurately the mutants showing substantial residual synthesis, and the possibility that these mutations are in a different gene(s) has not been excluded.A single mutant has been placed in a second group. Like some Group 1 mutants it synthesizes substantial amounts of DNA at 40° before synthesis stops. However, unlike them it supports the growth of T4 and Lambda at 41°. The DNA mutation maps near the leu locus. Certain properties of this mutant are consistent with the idea that initiation of DNA synthesis is temperature-sensitive in this strain.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported in part by U.S. Public Health Services Grant 5-TO1-GM00829 from the National Institute of General Medical Sciences and in part by U.S.P.H.S. research grant GM12524.