Biochemistry of DNA-Defective Amber Mutants of Bacteriophage T4 IV. DNA Synthesis in Plasmolyzed Cells

Requirements for bacteriophage T4 DNA synthesis have been investigated in situ by use of plasmolyzed infected cells. When such cells are incubated with dATP, dGTP, dTTP, hydroxymethyldeoxycytidine triphosphate, and rATP, significant semiconservative synthesis of DNA occurs. This DNA hybridizes preferentially to T4 DNA. T4 amber mutants defective in genes 44 and 45, which display a DNA-negative phenotype in vivo, are unable to synthesize DNA in situ. By contrast, T4 amber mutants bearing lesions in genes 41 and 62, which also display a DNA-negative phenotype in vivo, do allow DNA synthesis in situ, the extent of synthesis being 80 to 90% that of the wild-type synthesis under the same conditions. Cells infected with gene 42 mutants (dCMP hydroxymethylase) are unable to synthesize DNA in situ even though exogenous nucleotides are provided. Also one gene 1 mutant (deoxynucleotide kinase) was found to synthesize DNA in situ, but two other gene 1 mutants did not. These results point to possible roles of hydroxymethylase and kinase in DNA metabolism, in addition to provision of essential DNA precursors, as has recently been suggested by Wovcha et al. (1973).     

The bacteriophage P4 alpha gene is the structural gene for bacteriophage P4-induced RNA polymerase

Two temperature-sensitive mutants of satellite phage P4 which do not synthesize P4 DNA at the nonpermissive temperature have been isolated. One of these phage is mutated in the P4 alpha gene. It complements a P4 delta mutant, but not a P4 alpha amber mutant; both mutants are phenotypically identical to alpha amber mutants in all properties studied. They synthesize P4 early proteins 1 and 2 as well as two additional P4-induced early proteins, 5 and 6, which are described here. P4 late proteins are not synthesized by these mutants and cannot be transactivated by helper phage P2. The mutants are unable to transactivate P2 late proteins from a P2 AB mutant. The P4 RNA polymerase activity which has been suggested to be involved in P4 DNA synthesis is not detected at the nonpermissive temperature. The P4 polymerase activity in partially purified extracts prepared from cells infected with the mutant at the permissive temperature is temperature sensitive. Reduced activity is found in vitro when these extracts are preincubated at 41 degrees C or assayed at temperatures higher than 37 degrees C. Thus, the P4 RNA polymerase is the product of the alpha gene. Temperature shift experiments show that the alpha gene product is required until late in the P4 cycle.     

Isolation and preliminary characterization of amber mutants of bacteriophage phi W-14 defective in DNA synthesis.

Of 42 amber mutants of bacteriophage phi W-14, 6 were defective in DNA synthesis. Three of the mutants synthesized DNA in the nonpermissive host, but were defective in post-replicational modification of the DNA. The DNA synthesized by two of these mutants, am36 and am42, contained more thymine and less alpha-putrescinylthymine than did wild-type DNA; that synthesized by the third mutant, am37, contained the normal amount of thymine, no alpha-putrescinylthymine, and hydroxymethyluracil. The properties of these mutants suggested that the presence of the normal amount of alpha-putrescinylthymine in phi W-14 DNA was essential for the production of viable progeny. Three of the mutants, am6, am35, and am45, failed to synthesize DNA in the nonpermissive host. These mutants were analogous to the DNA off mutants of T4. Nonpermissive cells infected with DNA off mutants accumulated dATP, dGTP, dCTP, and hydroxymethyl dUTP, but not dTTP or alpha-putrescinyldeoxythymidine triphosphate, confirming that both thymine and alpha-putrescinylthymidine in phi W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level. The synthesis of phi W-14 DNA is unusual because (i) thymine is formed from hydroxymethyluracil at the polynucleotide level, (ii) the hypermodification forming alpha-putrescinylthymine is essential, and (iii) thymine and alpha-putrescinylthymine must be made in the correct proportions. Complementation tests showed that the mutants defined three genes involved in DNA polymerization and two genes involved in post-replicational modification.     

Influence of the growth rate on the macromolecular composition of Acinetobacter calcoaceticus in carbon-limited chemostat culture

Abstract Infectious phage particles can be formed in vitro when extracts of T1-infected cells are incubated with T1 DNA. The DNA packaging system is based on mixtures of complementing extracts from Escherichia coli sup⁰ cells infected with the amber mutants am 4 (gene 16) or am 10 (gene 13). Gene 16 mutants are defective in the formation of DNA-filled heads but make proheads; gene 13 mutants are defective in prohead formation. Three forms of DNA have been packaged: (1) endogenous concatemeric DNA present in mixtures of am 4 and am 10 mutant extracts; (2) concatemeric DNA; (3) virion DNA both when supplied exogenously to mixtures of am 4 · am 20 and am 10 · am 20 double mutant extracts ( am 20 inhibits T1 DNA synthesis). The reaction requires added ATP, Mg²⁺ and spermidine for optimum efficiency and produces about 1.5 × 10³ pfu/ μ g and about 1 × 10⁴ pfu/ μ g for exogenous concatemeric and virion DNA, respectively.     

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.     

Suppression of Amber Mutations of Bacteriophage T4 Gene 43 (DNA Polymerase) by Translational Ambiguity

The growth properties of twelve different amber (am) mutants of bacteriophage T4 gene 43 (DNA polymerase) were examined by using nonpermissive (su(-)) as well as permissive (su(+)) Escherichia coli hosts. It was found that most of these mutants were measurably suppressed in su(-) hosts by translational ambiguity (misreading of codons during protein synthesis). The ability of these mutants to grow in response to this form of weak suppression probably means that the T4 gene 43 DNA polymerase can be effective in supporting productive DNA replication when it is supplied in small amounts. By similar criteria, studies with other phage mutants suggested that the products of T4 genes 62 (uncharacterized), 44 (uncharacterized), 42 (dCMP-hydroxymethylase), and 56 (dCTPase) are also effective in small amounts. Some T4 gene products, such as the product of gene 41 (uncharacterized), seem to be partially dispensable for phage growth since am mutants of such genes do propagate, although weakly, in streptomycin-resistant su(-) hosts which appear to have lost the capacity to suppress am mutations by ambiguity.     

The extreme C terminus of herpes simplex virus DNA polymerase is crucial for functional interaction with processivity factor UL42 and for viral replication.

The herpes simplex virus DNA polymerase is composed of two subunits, a large catalytic subunit (Pol) and a smaller subunit (UL42) that increases the processivity of the holoenzyme. The interaction between the two polypeptides is of interest both for the mechanism by which it enables the enzyme to synthesize long stretches of DNA processively and as a possible target for the rational design of novel antiviral drugs. Here, we demonstrate through a combination of insertion and deletion mutagenesis that the carboxy-terminal 35 amino acids of Pol are crucial for binding UL42. The functional importance of the interaction was confirmed by the finding that a pol mutant defective for UL42 binding retained polymerase activity, but did not synthesize longer DNA products in the presence of UL42. Moreover, several association-incompetent mutants failed to complement the replication of a pol null mutant in a transient transfection assay, confirming that the Pol-UL42 interaction is necessary for virus replication in vivo and therefore a valid target for directed drug design.     

Topoisomerase II and other DNA-delay and DNA-arrest mutations impair bacteriophage T4 DNA packaging in vivo and in vitro.

A survey of DNA packaging in vivo and in vitro during infections caused by T4 DNA-delay and DNA-arrest amber mutants revealed a common DNA packaging-deficient phenotype. Electron microscopy revealed high proportions of proheads partially filled with DNA in vivo, indicating normal initiation but incomplete encapsidation. In contrast, exogenous mature T4 DNA was packaged in vitro by several early-gene mutant extracts. Detailed analysis of gene ts39 mutants (subunit of topoisomerase II) showed that in vivo packaging is defective, yet expression of late proteins appeared normal and the concatemeric DNA was not abnormally short or nicked. Although g39 amber mutant extracts packaged DNA in vitro, two of three ts39 mutant extracts prevented encapsidation of the exogenous DNA. The temperature-sensitive (ts) gp39 in a mutant topoisomerase II complex may have interfered with packaging in vivo and in vitro by interacting with DNA in an anomalous fashion, rendering it unfit for encapsidation. These results support the hypothesis that T4 DNA packaging is sensitive to DNA structure and discriminates against encapsidation of some types of defective DNA.     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

Non-essential UV-sensitive bacteriophage T4 mutants affecting early DNA synthesis: a third pathway of DNA repair.

Non-essential bacteriophage T4 mutants uvs58 and uvs79 showed a lower UV sensitivity than either the excision-repair mutant v am5 or the replication-dependent recombination-repair mutant y10. The UV sensitivity of double and triple mutants carrying one of the mutations uvs58 or uvs79, and v am 5 or (and) y10 was higher than the sum of the sensitivities of the single mutants. The uvs58 mutation was mapped to the early gene region, close to amN81 (gene 41). The unirradiated mutants uvs58 and uvs79 accumulated newly synthesized DNA at a slower rate than wild-type T4. Double mutants uvs58:am59 and uvs79:am59 showed DNA synthesis in E. coli B su- to be arrested at a 3--5 times lower level than that in am59-infected cells. Chloramphenicol, added 9--12 min after infection, suppressed arrests of DNA synthesis, the double mutants showing a lag of 8 min as compared with am59. Results from analysis of sucrose gradients of parental uvs58 and uvs79 DNA were in agreement with the suggestion of a mutation in an early function. The mutants uvs58 and uvs79 are suggested to be defective in a component of the DNA replication apparatus with a function in the adaptation to irregularities in the DNA structure. The third pathway of UV repair is tentatively designated as non-catalytic replication repair.     

On the Direction of Reading of Bacteriophage T4 Gene 43 (Deoxyribonucleic Acid Polymerase)

Amber (am) mutants of the two closely linked sites, B22 and C125, in bacteriophage T4 gene 43 [deoxyribonucleic acid (DNA) polymerase] synthesize in the nonpermissive (su(-)) Escherichia coli host gene 43 products which are devoid of DNA polymerase activity, but which retain a 3'-exonuclease activity. Diethylaminoethyl-cellulose chromatographic analysis of DNA polymerase and deoxyribonuclease activities from extracts of su(-) cells infected with single- and double-am mutants of T4 gene 43 showed that the exonuclease activity which is observed with amB22 is not seen with double mutants carrying, in addition to amB22, am mutations which map to the clockwise side of the B22 site on the circular genetic map of T4. Similarly, am mutations which map to the clockwise side of the C125 site abolish the exonuclease activity which is observed with an am mutant (amE4335) of this site. It was concluded that in these double mutants termination signals to the clockwise side of amB22 and amE4335 are encountered before the amB22 and amE4335 signals during translation of the messenger ribonucleic acid from T4 gene 43. Thus, it seems that the T4 DNA polymerase is synthesized in vivo in a direction which corresponds to a counterclockwise reading of gene 43.     

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.     

Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.     

Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks

ATR and Chk1 are protein kinases that perform major roles in the DNA replication checkpoint that delays entry into mitosis in response to DNA replication stress by hydroxyurea (HU) treatment. They are also activated by ionizing radiation (IR) that induces DNA double-strand breaks. Studies in human tissue culture and Xenopus egg extracts identified Claspin as a mediator that increased the activity of ATR toward Chk1. Because the in vivo functions of Claspin are not known, we generated Drosophila lines that each contained a mutated Claspin gene. Similar to the Drosophila mei-41/ATR and grp/Chk1 mutants, embryos of the Claspin mutant showed defects in checkpoint activation, which normally occurs in early embryogenesis in response to incomplete DNA replication. Additionally, Claspin mutant larvae were defective in G2 arrest after HU treatment; however, the defects were less severe than those of the mei-41/ATR and grp/Chk1 mutants. In contrast, IR-induced G2 arrest, which was severely defective in mei-41/ATR and grp/Chk1 mutants, occurred normally in the Claspin mutant. We also found that Claspin was phosphorylated in response to HU and IR treatment and a hyperphosphorylated form of Claspin was generated only after HU treatment in mei-41/ATR-dependent and tefu/ATM-independent way. In summary, our data suggest that Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks, and this difference is probably due to distinct phosphorylation statuses.     

A screen for Schizosaccharomyces pombe mutants defective in rereplication identifies new alleles of rad4+, cut9+ and psf2+

Fission yeast mutants defective in DNA replication have widely varying morphological phenotypes. We designed a screen for temperature-sensitive mutants defective in the process of replication regardless of morphology by isolating strains unable to rereplicate their DNA in the absence of cyclin B (Cdc13). Of the 42 rereplication-defective mutants analyzed, we were able to clone complementing plasmids for 10. This screen identified new alleles of the APC subunit cut9(+), the initiation/checkpoint factor rad4(+)/cut5(+), and the first mutant allele of psf2(+), a subunit of the novel GINS replication complex. Other genes identified are likely to play general roles in gene expression and protein localization.     

Studies on the biochemical basis of mutation VI. Selection and characterization of a new bacteriophage T4 mutator DNA polymerase

A new mutant of bacteriophage T4 has been isolated by a procedure which was designed to select for mutants with high spontaneous reversion rates. This mutant, M19, induces a defective DNA polymerase which has a degraded specificity and makes errors by inserting the incorrect nucleotide more frequently than the wild-type enzyme.In addition to M19, several other T4 polymerase amber and temperature-sensitive mutants have been located on a linear, fine-scale map. The mutants which most strongly affect mutation rates are found in two clusters at 25% and 80% of the gene. These two domains may represent the active site(s) of the polymerase and exonuclease activities.     

Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants.

To study the roles of viral genes in the establishment and maintenance of herpes simplex virus (HSV) latency, we have developed a polymerase chain reaction assay that is both quantitative and sensitive. Using this assay, we analyzed the levels of viral DNA in trigeminal ganglia of mice inoculated corneally with HSV mutants that are defective for virus replication at one or more sites in mice and for reactivation upon ganglionic explant. Ganglia from mice infected with thymidine kinase-negative mutants, which replicate at the site of inoculation and establish latency but do not replicate acutely in ganglia or reactivate upon explant, contained a range of levels of HSV DNA that overlapped with the range found in ganglia latently infected with wild-type virus. On average, these mutant-infected ganglia contained one copy of HSV DNA per 100 cell equivalents (ca. 10(4) molecules), which was 50-fold less than the average for wild-type virus. Ganglia from mice infected with a ribonucleotide reductase deletion mutant, which is defective for acute replication and reactivation upon ganglionic explant, also contained on average one copy of HSV DNA per 100 cell equivalents. We also detected substantial numbers of HSV DNA molecules (up to ca. 10(3] in ganglia of mice infected with an ICP4 deletion mutant and other replication-negative mutants that are severely impaired for viral DNA replication and gene expression. These results raise the possibility that such mutants can establish latency, which could have important implications for mechanisms of latency and for vaccine and antiviral drug development.     

Cellular transformation by adenovirus type 5 is influenced by the viral DNA polymerase.

Early region 2B (E2B) of the group C adenoviruses encodes a number of proteins, including the 140-kilodalton DNA polymerase, which plays a role in the initiation of viral DNA replication. Temperature-sensitive (ts) mutants with mutations mapping to E2B are conditionally defective for both DNA replication in human cells and transformation of rat cells. Nucleotide sequence analysis shows that the E2B mutant ts36 possesses a single point mutation specific to the viral DNA polymerase; this transition of a C to a T at position 7623 changes leucine residue 249 in the polymerase to a phenylalanine. A wild-type (ts+) revertant possesses a codon specifying the original leucine at position 249. Phenotypic analysis of revertant and wild-type viruses derived by marker rescue from ts36 shows that these variants are wild type for both viral DNA replication and transformation. Thus, the single point mutation in the polymerase gene of ts36 is responsible for both defects.     

In vivo functional interaction between DNA polymerase and dCMP-hydroxymethylase of bacteriophage T4.

Some mutations in the structural gene for T4 DNA polymerase (gene 43) behave as suppressors of a deficiency in T4 dCMP-hydroxymethylase (gene 42). The suppression appears to involve a functional interaction between the two enzymes at the level of DNA replication. The hydroxymethylase deficiency caused DNA structural abnormalities in replication, and DNA polymerase lesions appeared to partially reverse these abnormalities. The results do not necessarily imply protein-protein interactions between the two enzymes, although both enzymes appear to play roles in controlling the fidelity of phage DNA replication.