The mutA mistranslator tRNA-induced mutator phenotype requires recA and recB genes, but not the derepression of lexA-regulated functions

The mapping of mutA and mutC mutator alleles to the glyV and glyW glycine tRNA genes, respectively, and the subsequent discovery that the mutA phenotype is abolished in a DeltarecA strain raise the possibility that asp --> gly misinsertion may induce a novel mutagenic pathway. The recA requirement suggests three possibilities: (i) the SOS mutagenesis pathway is activated in mutA cells; (ii) loss of recA function interferes with mutA-promoted asp --> gly misinsertion; or (iii) a hitherto unrecognized recA-dependent mutagenic pathway is activated by translational stress. By assaying the expression levels of a reporter plasmid bearing a umuC :lacZ fusion, we show that the SOS regulon is not in a derepressed state in mutA cells. Neither overexpression of the lexA gene through a multicopy plasmid nor replacement of the wild-type lexA allele with the lexA1[Ind-] allele interferes with the expression of the mutA phenotype. The mutA phenotype is unaffected in cells defective for dinB, as shown here, and is unaffected in cells defective for umuD and umuC genes, as shown previously. We show that mutA-promoted asp --> gly misinsertion occurs in recA- cells and, therefore, the requirement for recA is 'downstream' of mistranslation. Finally, we show that the mutA phenotype is abolished in cells deficient for recB, suggesting that cellular recombination functions may be required for the expression of the mutator phenotype. We propose that translational stress induces a previously unrecognized mutagenic pathway in Escherichia coli.     

Requirement for homologous recombination functions for expression of the mutA mistranslator tRNA-induced mutator phenotype in Escherichia coli

Expression of the Escherichia coli mutA mutator phenotype requires recA, recB, recC, ruvA, and ruvC gene, but not recD, recF, recO, or recR genes. Thus, the recBCD-dependent homologous recombination system is a component of the signal pathway that activates an error-prone DNA polymerase in mutA cells.     

DNA polymerase III from Escherichia coli cells expressing mutA mistranslator tRNA is error-prone

Translational stress-induced mutagenesis (TSM) refers to the elevated mutagenesis observed in Escherichia coli cells in which mistranslation has been increased as a result of mutations in tRNA genes (such as mutA) or by exposure to streptomycin. TSM does not require lexA-regulated SOS functions but is suppressed in cells defective for homologous recombination genes. Crude cell-free extracts from TSM-induced E. coli strains express an error-prone DNA polymerase. To determine whether DNA polymerase III is involved in the TSM phenotype, we first asked if the phenotype is expressed in cells defective for all four of the non-replicative DNA polymerases, namely polymerase I, II, IV, and V. By using a colony papillation assay based on the reversion of a lacZ mutant, we show that the TSM phenotype is expressed in such cells. Second, we asked if pol III from TSM-induced cells is error-prone. By purifying DNA polymerase III* from TSM-induced and control cells, and by testing its fidelity on templates bearing 3,N(4)-ethenocytosine (a mutagenic DNA lesion), as well as on undamaged DNA templates, we show here that polymerase III* purified from mutA cells is error-prone as compared with that from control cells. These findings suggest that DNA polymerase III is modified in TSM-induced cells.     

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.     

The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --> A mutations predominating over C --> T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.     

Fidelity of uracil-initiated base excision DNA repair in DNA polymerase beta-proficient and -deficient mouse embryonic fibroblast cell extracts

Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase beta (+/+) and (-/-) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U.A, U.G, and U.T targets contained within the lacZalpha gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase beta (+/+) and (-/-) cell-free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced ( approximately 88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting approximately 20% of the repair events. Addition of p21 peptide or protein to DNA polymerase beta (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by approximately 2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U.T) DNA was determined to be 5.7-7.2 x 10(-4) when using DNA polymerase beta (+/+) and (-/-) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U.G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U.G target and frequently involved a 1-base deletion or incorporation of dTMP.     

Specificity of spontaneous mutations induced in mutA mutator cells

Escherichia coli cells expressing the mutA allele of a glyV (glycine tRNA) gene express a strong mutator phenotype. The mutA allele differs from the wild type glyV gene by a base substitution in the anticodon such that the resulting tRNA misreads certain aspartate codons as glycine, resulting in random, low-level Asp-->Gly substitutions in proteins. Subsequent work showed that many types of mistranslation can lead to a very similar phenotype, named TSM for translational stress-induced mutagenesis. Here, we have determined the specificity of forward mutations occurring in the lacI gene in mutA cells as well as in wild type cells. Our results show that in comparison to wild type cells, base substitutions are elevated 23-fold in mutA cells, as against a eight-fold increase in insertions and a five-fold increase in deletions. Among base substitutions, transitions are elevated 13-fold, with both G:C-->A:T and A:T-->G:C mutations showing roughly similar increases. Transversions are elevated 35-fold, with G:C-->T:A, G:C-->C:G and A:T-->C:G elevated 28-, 13- and 27-fold, respectively. A:T-->T:A mutations increase a striking 348-fold over parental cells, with most occurring at two hotspot sequences that share the G:C-rich sequence 5'-CCGCGTGG. The increase in transversion mutations is similar to that observed in cells defective for dnaQ, the gene encoding the proofreading function of DNA polymerase III. In particular, the relative proportions and sites of occurrence of A:T-->T:A transversions are similar in mutA and mutD5 (an allele of dnaQ) cells. Interestingly, transversions are also the predominant base substitutions induced in dnaE173 cells in which a missense mutation in the alpha subunit of polymerase III abolishes proofreading without affecting the 3'-->5' exonuclease activity of the epsilon subunit.     

Mechanisms of mutagenesis by exocyclic DNA adducts. Transfection of M13 viral DNA bearing a site-specific adduct shows that ethenocytosine is a highly efficient RecA-independent mutagenic noninstructional lesion

It is widely accepted that mutagenic DNA lesions fall into two categories: mispairing lesions hydrogen bond with an incorrect incoming base, generally do not stop replication, and possess high mutagenic efficiency without any requirement for induced functions; noninstructional lesions lack accessible template information, act as strong blocks to DNA replication (and are therefore toxic), and their mutagenic effects are SOS-dependent. Our recent results show that ethenocytosine (epsilon C), a noninstructional exocyclic DNA lesion induced by vinyl chloride, may have unusual mutagenic properties. To obtain more definitive experimental evidence for the observed effects, we have introduced a single epsilon C residue at a specific site of coliphage M13AB28 replicative form DNA by a "single-stranded linker-ligation" technique. The resulting DNA was purified and transfected into appropriate recA+ or recA- Escherichia coli host cells. The effect of epsilon C on survival was determined from transfection efficiency. Both the frequency and specificity of mutations induced by epsilon C were determined by direct sequence analysis of randomly picked progeny phage plaques. The results indicated that epsilon C has little effect on the survival of M13 DNA. Approximately 30% of the progeny phage obtained by transfecting epsilon C DNA had a base substitution mutation precisely at the lesion site. No such mutations were observed in progeny plaques obtained by transfecting the control DNA construct. All epsilon C-induced mutations were either C-to-T transitions or C-to-A transversions. Neither survival nor mutagenic efficiency was significantly affected in recA- host cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli . The recombinant protein bound to the promoter regions of the M. leprae lexA , M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis . Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis . Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti- M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.     

Quantification of RecA protein in Deinococcus radiodurans reveals involvement of RecA,but not LexA,in its regulation

RecA protein is essential for the very high level of resistance of Deinococcus radiodurans to DNA damage induced by ionizing radiation or other DNA-damaging agents. Since the mechanism(s) involved in the control of recA expression and the extent of RecA induction following DNA damage in this species are still unclear, we have performed a genetic analysis of the recA locus and quantified the basal and induced levels of RecA protein in wild type, recA, and lexA mutants. We found that the two genes upstream of recA in the predicted cinA ligT recA operon appear to have no role in the regulation of recA expression or function, despite the fact that the reading frames in the operon overlap. By using a translational fusion of recA to a lacZ reporter gene, we showed that induction began with no delay following exposure to gamma-radiation or treatment with mitomycin, and continued at a constant rate until it reached a plateau. The induction efficiency increased linearly with inducer dose, levelling off at a concentration fourfold above the background. The basal concentration of RecA protein measured by Western blotting corresponded to approximately 11,000 monomers per cell, and the induced concentration to around 44,000 monomers per cell. These levels remained unchanged upon disruption of the lexA gene, indicating that LexA does not plays a role in recA regulation. However, inactivation of lexA caused cells to aggregate, suggesting that LexA may control the activity or expression of as yet undefined membrane functions. Cells bearing the recA670 mutation showed an elevated constitutive expression of recA in the absence of DNA damage. This phenotype did not result from the defect in DNA repair associated with the RecA670 protein, since the increased basal level of recA expression was also found in recA670/ recA(+) diploid cells that are proficient in DNA repair. These results suggest that RecA may be involved in regulating its own expression, possibly by stimulating proteolytic modification of other regulatory proteins.     

Mutation rate is reduced by increased dosage of mutL gene in Escherichia coli K-12

A variable but substantial proportion of wild Escherichia coli isolates present consistently lower mutation frequencies than that found in the ensemble of strains. The genetic mechanisms responsible for the hypo-mutation phenotype are much less known than those involved in hyper-mutation. Changes in E. coli mutation frequencies derived from the gene-copy effect of mutS, mutL, mutH, uvrD, mutT, mutY, mutM, mutA, dnaE, dnaQ, and rpoS are explored. When present in a very high copy number ( approximately 300 copies cell(-1)), mutL, mutH, and mutA gene copies yielded >/=twofold decrease in mutation rates determined by Luria-Delbrück fluctuation tests. Nevertheless, when the copy number was not such high ( approximately 15 copies cell(-1)), only mutL results in a consistent twofold decrease in the mutation rate. This reduction seems to be independent from the RecA background, phase of growth, or from the presence of proficient MutS. An increase in mutL gene copies was also able to partially compensate the hypermutator phenotype of a mutS-defective E. coli derivative.     

Conditional lethality of the recA441 and recA730 mutants of Escherichia coli deficient in DNA polymerase I

E. coli strains bearing the recA441 mutation and various mutations in the polA gene resulting in enzymatically well-defined deficiencies of DNA polymerase I have been constructed. It was found that the recA441 strains bearing either the polA1 or polA12 mutation causing deficiency of the polymerase activity of pol I are unable to grow at 42 degrees C on minimal medium supplemented with adenine, i.e., when the SOS response is continuously induced in strains bearing the recA441 mutation. Under these conditions the inhibition of DNA synthesis is followed in recA441 polA12 by DNA degradation and loss of cell viability. A similar lethal effect is observed with the recA730 polA12 mutant. The recA441 strain bearing the polA107 mutation resulting in the deficiency of the 5'-3' exonuclease activity of pol I shows normal growth under conditions of continuous SOS response. We postulate that constitutive expression of the SOS response leads to an altered requirement for the polymerase activity of pol I.     

Alkylating Agents Induce Uvm, a Reca-Independent Inducible Mutagenic Phenomenon in Escherichia Coli

Noninstructive DNA damage in Escherichia coli induces SOS functions hypothesized to be required for mutagenesis and translesion DNA synthesis at noncoding DNA lesions. We have recently demonstrated that in E. coli cells incapable of SOS induction, prior UV-irradiation nevertheless strongly enhances mutagenesis at a noninstructive lesion borne on M13 DNA. Here, we address the question whether this effect, named UVM for UV modulation of mutagenesis, can be induced by other DNA damaging agents. Exponentially growing δrecA cells were pretreated with alkylating agents before transfection with M13 single-stranded DNA bearing a site-specific ethenocytosine residue. Effect of cell pretreatment on survival of the transfected DNA was determined as transfection efficiency. Mutagenesis at the ethenocytosine site in pretreated or untreated cells was analyzed by multiplex DNA sequencing, a phenotype-independent technology. Our data show that 1-methyl-3-nitro-1-nitrosoguanidine, N-nitroso-N-methylurea and dimethylsulfate, but not methyl iodide, are potent inducers of UVM. Because alkylating agents induce the adaptive response to defend against DNA alkylation, we asked if the genes constituting the adaptive response are required for UVM. Our data show that MNNG induction of UVM is independent of ada, alkA and alkB genes and define UVM as an inducible mutagenic phenomenon distinct from the E. coli adaptive and SOS responses.     

Can tRNAs act as antisense RNA? The case of mutA and dnaQ

Many mutator genes have been characterized in E. coli, but the realization that mutA, the most recent mutator pathway described, encodes for a missense suppressor glycine tRNA caused a real surprise. The connection between expression of mutA and a 10 times increase in the spontaneous mutation rate is not readily explainable. The first attempt to describe the mechanism of action suggested a direct mistranslation of one subunit of polymerase III (PolIII) and the ideal candidate was the epsilon subunit carrying the 3'-->5' exonuclease activity. This subunit increases PolIII accuracy about 100 times. However, such direct mistranslation of epsilon was later ruled out when it became clear that all mutA cells express an error-prone form of PolIII. This result could not be reconciled with the very low level of mistranslation (1%) caused by mutA. But there is no need to invoke amino acid misincorporation in epsilon to destroy its activity. On the contrary, I suggest a new way to regulate epsilon amount, based on the reinterpretation of the mutA pathway through the new and puzzling observation that several tRNAs (including mutA which encodes for a glycine missense suppressor tRNA) are complementary to the 5' end of dnaQ mRNA. Accordingly, I propose that uncharged tRNAs can act as antisense RNAs, decreasing translation of dnaQ and possibly other genes. This could represent a new regulatory function for tRNAs and of course gives a direct and unrecognized link between starvation and mutation rate.     

Replication bypass and mutagenic effect of alpha-deoxyadenosine site-specifically incorporated into single-stranded vectors.

alpha-2'-Deoxyadenosine (alpha) is a major adenine lesion produced by gamma-ray irradiation of DNA under anoxic conditions. In this study, single-stranded recombinant M13 vectors containing alpha were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion. The data for alpha were further compared with those obtained with M13 vectors containing normal A or a model abasic site (F) at the same site. The transfection assay revealed that alpha constituted a moderate block to DNA replication. The in vivo replication capacity to pass through alpha was approximately 20% relative to normal A, but 20-fold higher than that of F constituting an almost absolute replication block. Similar data were obtained by in vitro replication of oligonucleotide templates containing alpha or F by E.coli DNA polymerase I. The mutagenic consequence of replicating M13 DNA containing alpha was analyzed by direct DNA sequencing of progeny phage. Mutagenesis was totally targeted at the site of alpha introduced into the vector. Mutation was exclusively a single nucleotide deletion and no base substitutions were detected. The deletion frequency associated alpha was dependent on the 3'-nearest neighbor base: with the 3'-nearest neighbor base T mutation (deletion) frequency was 26%, whereas 1% with the 3'-nearest neighbor base G. A possible mechanism of the single nucleotide deletion associated with alpha is discussed on the basis of the misinsertion-strand slippage model.     

Characterization of DNA polymerase I*, a form of DNA polymerase I found in Escherichia coli expressing SOS functions

DNA polymerase I* is a form of the DNA polymerase I isolated from Escherichia coli which are expressing recA/lexA (SOS) functions. Induction of recA or polA1 cells by nalidixic acid does not result in the appearance of pol I*, but lexA or recA mutants that are constitutive for SOS functions constitutively express pol I* and mutants which lack functional recA protein produce pol I* when they carry a lexA mutation which renders the lexA repressor inoperative. Pol I* has been induced by nalidixic acid in dinA, dinD, dinF, and umuC mutants. Polymerase I* has a lower affinity for single-stranded DNA-agarose than polymerase I and it sediments through sucrose gradients in a dispersed manner between 6.6-10.5 S, whereas polymerase I sediments at 5 S. Whereas pol I* migrates significantly faster than pol I in nondenaturing polyacrylamide gels, the active polypeptide of both forms migrates at the same rate in denaturing polyacrylamide gels. Compared with polymerase I, polymerase I* has an enhanced capacity to incorporate the adenine analog, 2-amino-purine, into activated salmon sperm DNA and a relatively low fidelity in replicating synthetic polydeoxyribonucleotides. Both the 3'----5' (proofreading) and 5'----3' (nick-translational) exonuclease activities of pol I* and pol I are indistinguishable. Estimates of processivity give a value of approximately 6 for both forms of the enzyme.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase.

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.     

Rates of Spontaneous Mutation in Bacteriophage T4 Are Independent of Host Fidelity Determinants

Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity.