In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

Cold-sensitive rad52 alleles of yeast

We have characterized two rad52 mutations that are cold-sensitive for growth on MMS agar. The mutations change residues 61 and 69, respectively, in the 504 amino-acid open reading frame. Neither mutation has a profound effect on mitotic crossing-over or on gene conversion. One has a severe deficiency in sporulation at all temperatures, while the other has a partial deficiency and reduced spore viability. Both mutants are retarded in growth on MMS agar by a high-copy plasmid expressing RAD51. Received: 18 April / 14 May 1997     

The repair of DNA methylation damage in Saccharomyces cerevisiae

 The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways. Received: 13 June/24 July 1996     

Arabidopsis thaliana RAD6 homolog AtUBC2 complements UV sensitivity, but not N-end rule degradation deficiency, of Saccharomyces cerevisiae rad6 mutants

AtUBC2 of Arabidopsis thaliana encodes a structural homolog of the RAD6 gene of Saccharomyces cerevisiae with approximately 65% identical amino acids. Like structural homologs from other organisms, AtUBC2 lacks the carboxyl-terminal extension of mostly acidic amino acids which is present in Rad6p. AtUBC2 was expressed in S. cerevisiae rad6 mutants. It was found to partially complement the UV sensitivity and reduced growth rate of rad6 mutants at elevated temperatures. AtUBC2 however, has no apparent influence on the degradation of N-end rule substrates in the heterologous host. Received: 7 July 1997 / 27 August 1997     

Human and mouse homologs of Schizosaccharomyces pombe rad1+ and Saccharomyces cerevisiae RAD17: linkage to checkpoint control and mammalian meiosis

Preventing or delaying progress through the cell cycle in response to DNA damage is crucial for eukaryotic cells to allow the damage to be repaired and not incorporated irrevocably into daughter cells. Several genes involved in this process have been discovered in fission and budding yeast. Here, we report the identification of human and mouse homologs of the Schizosaccharomyces pombe DNA damage checkpoint control gene rad1⁺ and its Saccharomyces cerevisiae homolog RAD17. The human gene HRAD1 is located on chromosome 5p13 and is most homologous to S. pombe rad1⁺. This gene encodes a 382-amino-acid residue protein that is localized mainly in the nucleus and is expressed at high levels in proliferative tissues. This human gene significantly complements the sensitivity to UV light of a S. pombe strain mutated in rad1⁺. Moreover, HRAD1 complements the checkpoint control defect of this strain after UV exposure. In addition to functioning in DNA repair checkpoints, S. cerevisiae RAD17 plays a role during meiosis to prevent progress through prophase I when recombination is interrupted. Consistent with a similar role in mammals, Rad1 protein is abundant in testis, and is associated with both synapsed and unsynapsed chromosomes during meiotic prophase I of spermatogenesis, with a staining pattern distinct from that of the recombination proteins Rad51 and Dmc1. Together, these data imply an important role for hRad1 both in the mitotic DNA damage checkpoint and in meiotic checkpoint mechanisms, and suggest that these events are highly conserved from yeast to humans.     

Genetic interactions between mutants of the `error-prone' repair group of Saccharomyces cerevisiae and their effect on recombination and mutagenesis

We have created an isogenic series of yeast strains that carry genetic systems to monitor different types of recombination and mutation [B. Liefshitz, A. Parket, R. Maya, M. Kupiec, The role of DNA repair genes in recombination between repeated sequences in yeast, Genetics 140 (1995) 1199–1211.]. In the present study we characterize the effect of mutations in genes of the `error-prone' or postreplicative repair group on recombination and mutation. We show that rad5 and rad18 strains have elevated levels of spontaneous recombination, both of ectopic gene conversion and of recombination between direct repeats. The increase in recombination levels is similar in both mutants and in the rad5 rad18 double mutant, suggesting that the RAD5 and RAD18 gene products act together with respect to spontaneous recombination. In contrast, RAD5 and RAD18 play alternative roles in mutagenic repair: mutations in each of these genes elevate spontaneous forward mutation at the CAN1 locus, but when both genes are deleted, a low level of spontaneous mutagenesis is seen. The RAD5/RAD18 pathway of mutagenic repair is dependent on the REV3-encoded translesion polymerase. We analyze the interactions between the RAD5 and RAD18 gene products and other repair genes. The high recombination levels seen in rad5 and rad18 mutants is dependent on the RAD1, RAD51, RAD52, and RAD57 genes. The Srs2 helicase plays an important role in creating the recombinogenic substrate(s) processed by the RAD5 and RAD18 gene products.     

Homologous recombination in the fission yeast Schizosaccharomyces pombe: different requirements for the rhp51+, rhp54+ and rad22+ genes

The Schizosaccharomyces pombe rhp51 ⁺ , rad22 ⁺ and rhp54 ⁺ genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Δ and rhp54Δ strains are extremely sensitive to ionizing radiation; the rad22Δ mutant turned out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Δ and rhp54Δ are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Δ mutant is only reduced by a factor of two. Blot-analysis demonstrated that the majority of the leu⁺ transformants of the wild-type and rad22Δ strains have integrated one or more copies of the vector. Gene conversion events were observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Δ and a rhp54Δ background. Meiotic recombination is hardly affected in the rad22Δ mutant. The rhp51Δ and rhp54Δ strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Δ strain and 27% in the rhp54Δ strain, as compared with wild-type cells. However, in the rhp51Δ mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe. Received: 20 July 1996     

The HMG-domain protein Ixr1 blocks excision repair of cisplatin-DNA adducts in yeast

Ixr1 is a yeast HMG-domain protein which binds the major DNA adducts of the antitumor drug cisplatin. Previous work demonstrated that Saccharomyces cerevisiae cells lacking the IXR1 gene were two-fold less sensitive to cisplatin treatment than wild-type cells, and the present investigation reveals a six-fold difference in yeast having a different background. The possibility that the lower cytotoxicity of cisplatin in the ixrl strain is the result of enhanced repair was investigated in rad1, rad2, rad4, rad6, rad9, rad10, rad14 and rad52 backgrounds. In three of the excision repair mutants, rad2, rad4 and rad14, the differential sensitivity caused by removing the Ixr1 protein was nearly abolished. This result demonstrates that the greater cisplatin resistance in the ixrl strain is most likely a consequence of excision repair, supporting the theory that Ixr1 and other HMG-domain proteins can block repair of the major cisplatin-DNA adducts in vivo. The differential sensitivity of wild-type cells and those lacking Ixr1 persisted in the rad1 and rad10 strains, however, indicating that these two proteins act at a stage in the excision repair pathway where damage recognition is less critical. A model is proposed to account for these results, which is strongly supported recently identified functional roles for the rad excision repair gene products. A rad52 mutant was more sensitive to cisplatin than the RAD52 parental strain, which reveals that Rad52, a double-strand break repair protein, repairs cisplatin-DNA adducts, probably interstrand cross-links. A rad52 ixrl strain was less sensitive to cisplatin than the rad52 IXR1 strain, consistent with Ixr1 not blocking repair of cisplatin adducts removed by Rad52, rad6 strains behaved similarly, except they were both substantially more sensitive to cisplatin. Interruption of the RAD9 gene, which is involved in DNA-damage-induced cell cycle arrest, had no affect on cisplatin cytotoxicity.     

The pattern of sensitivity of yeast dna2 mutants to DNA damaging agents suggests a role in DSB and postreplication repair pathways

The Saccharomyces cerevisiae DNA2 gene encodes a DNA-stimulated ATPase and DNA helicase/nuclease essential for DNA replication. In characterizing dna2 mutants, we have found that Dna2p also participates in DNA repair or in damage avoidance mechanisms. dna2 mutants are sensitive to X rays, although they are less sensitive than rad52 mutants. The X-ray sensitivity of dna2 mutants is suppressed by overexpression of a 5′ to 3′ exonuclease, the yeast FEN-1 structure-specific nuclease, encoded by the RAD27 gene, which also suppresses the growth defect of dna2-ts mutants. SGS1 encodes a helicase with similar properties to Dna2 protein. Although sgs1Δ mutants are resistant to X rays, dna2-2 sgs1Δ double mutants are more sensitive to X rays than the dna2-2 mutant. Temperature sensitive dna2 mutants are only slightly sensitive to UV light, show normal levels of spontaneous and UV induced mutagenesis, and have only a 2.5-fold elevated level of dinucleotide tract instability compared to wildtype. However, dna2Δ strains kept alive by overproduction of RAD27 are highly sensitive to UV light. These phenotypes, in addition to the epistasis analysis reported, allow us to propose that Dna2 is involved in postreplication and DSB repair pathways.     

CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint

Initiation of DNA replication during the mitotic cell cycle requires the activation of a cyclin-dependent protein kinase (CDK). The B-type cyclins Clb5 and Clb6 are the primary activators of the S phase function of the budding yeast CDK Cdc28. However, in mitotically growing cells this role can be fulfilled by the other B-type cyclins Clb1–Clb4. We report here that cells undergoing meiotic development also require Clb dependent CDK activity for DNA replication. Diploid clb5/clb5 clb6/clb6 mutants are unable to perform premeiotic DNA replication. Despite this defect, the mutant cells progress into the meiotic program and undergo lethal segregation of unreplicated DNA suggesting that they fail to activate a checkpoint that restrains meiotic M phase until DNA replication is complete. We have found that a DNA replication checkpoint dependent on the ATM homolog MEC1 operates in wild-type cells during meiosis and can be invoked in response to inhibition of DNA synthesis. Although cells that lack clb5 and clb6 are unable to activate the meiotic DNA replication checkpoint, they do possess an intact DNA damage checkpoint which can restrain chromosome segregation in the face of DNA damage. We conclude that CLB5 and CLB6 are essential for premeiotic DNA replication and, consequently, for activation of a meiotic DNA replication checkpoint.     

Rad18/Rad5/Mms2-mediated polyubiquitination of PCNA is implicated in replication completion during replication stress

Ubiquitination of proteins was previously shown to modulate various processes of DNA metabolism. PCNA, a processivity factor with essential functions in replication and repair, is modified with ubiquitin at K164. In addition, PCNA is sumoylated at K127 and K164. We found that the rad18delta mutation suppresses the temperature sensitivity of the polymerase delta mutants hys2-1 and cdc2-1 as well as the synthetic lethality of cdc2-1 pol32delta mutants, suggesting a role for Rad18 in modulating DNA replication. As Rad18 mediates ubiquitination of PCNA, we examined whether PCNA modifications affected its function in replication. Multicopy PCNA alleviated the replication defects of rfc5-1 strains, but not those of poldelta mutants. In contrast, multicopy PCNA-K164R had reduced ability to suppress the replication defects of rfc5-1, but alleviated those of poldelta mutants. The roles of sumoylated and ubiquitinated PCNA in rfc5-1 and hys2-1 mutants were addressed by using mutant backgrounds that selectively affected sumoylation (siz1delta), ubiquitination (rad18delta), polyubiquitination (rad5delta, mms2delta), or the ability of cells to perform translesion synthesis (polzetadelta, poletadelta). Our results are consistent with the idea that the Rad18/Rad5/Mms2 polyubiquitination pathway is important for replication completion, perhaps by promoting a template switch type of DNA synthesis.     

Determination of hprt mutant and mutation frequencies and the molecular characterization of human derived in vivo T-lymphocyte mutants

Using a T-lymphocyte clonal assay, 73 6-thiogua-nine resistant T-lymphocytes were isolated from two blood samples obtained 4 months apart from a 50-year-old male subject. Sixty-six of these mutants were characterized at the DNA sequence level using cDNA. One particular single base substitution was recovered a total of 23 times. The majority of T-cell receptors (TCR) of these mutants all share a common γ-TCR rearrangement, and thus likely represent a single mutational event that underwent clonal expansion in vivo. Siblings of this clone were recovered in both collections. Three other single base substitutions were also recovered more than once. In two of the three cases, the mutants were also found to be clonally related, while in one case they were not. A number of identical exon loss events were also recovered, yet none of these were clonally related. This probably reflects the multiple pathways by which these mutations can arise. The TCR data was used to correct the observed mutant frequency to produce an estimate of the actual mutation frequency. The two mutant frequencies, 18 × 10^?6 and 19 × 10^?6, obtained from the first and second sampling periods, respectively, can thus be corrected to yield true mutation frequency's of 12 × 10^?6 each © 1995 Wiley-Liss, Inc.     

Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans

DNA lesions that block replication fork progression are drivers of cancer-associated genome alterations, but the error-prone DNA repair mechanisms acting on collapsed replication are incompletely understood, and their contribution to genome evolution largely unexplored. Here, through whole-genome sequencing of animal populations that were clonally propagated for more than 50 generations, we identify a distinct class of deletions that spontaneously accumulate in C. elegans strains lacking translesion synthesis (TLS) polymerases. Emerging DNA double-strand breaks are repaired via an error-prone mechanism in which the outermost nucleotide of one end serves to prime DNA synthesis on the other end. This pathway critically depends on the A-family polymerase theta, which protects the genome against gross chromosomal rearrangements. By comparing the genomes of isolates of C. elegans from different geographical regions, we found that in fact most spontaneously evolving structural variations match the signature of polymerase theta-mediated end joining (TMEJ), illustrating that this pathway is an important source of genetic diversification.Identifying the mechanisms that fuel genome change is crucial for understanding evolution and carcinogenesis. Spontaneous mutagenesis is caused predominantly by misinsertions or slippage events of replicative polymerases that are missed by their proofreading domains and not corrected by mismatch repair (Lynch 2008). Less frequently, but with a potentially much more detrimental effect, mutations can arise when DNA damage obstructs progression of DNA replication; and stalled replication forks eventually collapse, resulting in highly mutagenic double-stranded breaks (DSBs). Although error-free homologous repair, in which the sister chromatid is used as a template, restores the original sequence, infrequent but highly mutagenic error-prone end joining processes can give rise to spontaneous deletions and tumor-promoting translocations (Mitelman et al. 2007).To circumvent fork collapse at DNA damage, cells use various alternative polymerases that are capable of incorporating nucleotides across DNA lesions and are hence called translesion synthesis (TLS) polymerases. TLS acts on a wide variety of DNA lesions that can result from endogenous as well as exogenous genotoxic sources: DNA lesions that result from UV-light exposure, for instance, are efficiently bypassed by the well-conserved TLS polymerase eta (pol eta), inactivation of which in humans leads to the variant form of the skin cancer predisposition syndrome, Xeroderma Pigmentosum (Masutani et al. 1999b; Johnson et al. 2007). Abundant in vitro studies demonstrate the involvement of TLS polymerases pol eta and pol kappa in the bypass of lesions that are produced by endogenous reactive compounds, showing that these polymerases are also essential for protection of the genome under unchallenged conditions (Haracska et al. 2000; Fischhaber et al. 2002; Kusumoto et al. 2002).Although error-prone while replicating, and thus potentially causing misinsertions, TLS polymerases are thought to protect cells against the more mutagenic effects of replication fork collapse (Knobel and Marti 2011). Here, we investigate the contribution of TLS polymerases on the maintenance of genome stability and the mechanisms acting on stalled DNA replication by characterizing C. elegans strains that are defective for the Y-family polymerases pol eta and pol kappa. Unexpectedly, we found that DSBs resulting from replication-blocking endogenous lesions are not repaired via canonical DSB repair pathways, but through an error-prone repair mechanism that critically depends on the A-family DNA polymerase theta (pol theta).     

MglA and Igl proteins contribute to the modulation of Francisella tularensis live vaccine strain-containing phagosomes in murine macrophages

The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.     

Virulence and karyotype analyses of rad52 mutants of Candida albicans: regeneration of a truncated chromosome of a reintegrant strain (rad52/RAD52) in the host

The virulence of Candida albicans mutants lacking one or both copies of RAD52, a gene involved in homologous recombination (HR), was evaluated in a murine model of hematogenously disseminated candidiasis. In this study, the virulence of the rad52Delta mutant was dependent upon the inoculum concentration. Mice survived at a cell inoculum of 1 x 10(6), but there was a decrease in survival time at dosages of 1.5 x 10(6) and especially at 3 x 10(6) cells per animal. The heterozygote RAD52/rad52 behaved like wild type, whereas a reintegrant strain was intermediate in its ability to cause death compared to these strains and to the avirulent rad52/rad52 null at inocula of 1 x 10(6) and 1.5 x 10(6) cells. A double mutant, lig4/lig4/rad52/rad52, was avirulent at all inocula used. PCR analysis of the RAD52 and/or LIG4 loci showed that all strains recovered from animals matched the genotype of the inoculated strains. Analysis of the electrophoretical karyotypes indicated that the inoculated, reintegrant strain carried a large deletion in one copy of chromosome 6 (the shortest homologue, or Chr6b). Interestingly, truncated Chr6b was regenerated in all the strains recovered from moribund animals using the homologue as a template. Further, regeneration of Chr6b was paralleled by an increase in virulence that was still lower than that of wild type, likely because of the persistent loss of heterozygosity in the regenerated region. Overall, our results indicate that systemic candidiasis can develop in the absence of HR, but simultaneous elimination of both recombination pathways, HR and nonhomologous end-joining, suppresses virulence even at very high inocula.     

Adenovirus early region 1b gene function required for rescue of latent adeno-associated virus

Various early and late temperature-sensitive mutants of adenovirus (Ad) were able to support adeno-associated virus (AAV) DNA replication in either coinfections or in the rescue of AAV from latently infected human (Detroit 6) cells. Group I Ad host range mutants (hr 3, early region 1a) were unable to help AAV DNA replication in either case. Group II Ad host range mutants (hr 6, early region 1b), however, were able to help AAV DNA replication in coinfection but were unable to rescue AAV DNA replication in latently infected Detroit 6 cells. Thus, an Ad function(s), in addition to those required for AAV DNA replication, is needed for AAV rescue.     

Interactions among genes controlling sensitivity to radiation (RAD) and to alkylation by nitrogen mustard (SNM) in yeast

Summary Three haploid yeast mutants (snm) sensitive or thermoconditionally sensitive to the DNA cross-linking agent nitrogen mustard (HN2) were crossed with four rad strains representing mutations in the three pathways of DNA dark repair. The resulting haploid double and triple mutant strains were tested for their sensitivity to UV, HN2 and HN1. From the observed epistatic or synergistic interactions of the combinations of mutant alleles we could derive the relation of the SNM1 and SNM2 genes to the postulated repair pathways. Alleles snm1-1 and snml-2 ^ts were found epistatic to genes of the rad3 group, whereas snm2-1 ^ts was epistatic to rad6. The snm1 and snm2 mutant alleles interacted synergistically. From these data it is concluded that the SNM1 gene product plays a cross-link specific role in excision repair while the SNM2 gene product may be involved in a system of error-prone repair.     

Hyphal differentiation induced via a DNA damage checkpoint-dependent pathway engaged in crosstalk with nutrient stress signaling in Schizosaccharomyces japonicus

DNA damage response includes DNA repair, nucleotide metabolism and even a control of cell fates including differentiation, cell death pathway or some combination of these. The responses to DNA damage differ from species to species. Here we aim to delineate the checkpoint pathway in the dimorphic fission yeast Schizosaccharomyces japonicus, where DNA damage can trigger a differentiation pathway that is a switch from a bidirectional yeast growth mode to an apical hyphal growth mode, and the switching is regulated via a checkpoint kinase, Chk1. This Chk1-dependent switch to hyphal growth is activated with even low doses of agents that damage DNA; therefore, we reasoned that this switch may depend on other genes orthologous to the components of the classical Sz. pombe Chk1-dependent DNA checkpoint pathway. As an initial test of this hypothesis, we assessed the effects of mutations in Sz. japonicus orthologs of Sz. pombe checkpoint genes on this switch from bidirectional to hyphal growth. The same set of DNA checkpoint genes was confirmed in Sz. japonicus. We tested the effect of each DNA checkpoint mutants on hyphal differentiation by DNA damage. We found that the Sz. japonicus hyphal differentiation pathway was dependent on Sz. japonicus orthologs of Sz. pombe checkpoint genes—_SP rad3, _SP rad26, _SP rad9, _SP rad1, _SP rad24, _SP rad25, _SP crb2, and _SP chk1—that function in the DNA damage checkpoint pathway, but was not dependent on orthologs of two Sz. pombe genes—_SP cds1 or _SP mrc1—that function in the DNA replication checkpoint pathway. These findings indicated that although the role of each component of the DNA damage checkpoint and DNA replication checkpoint is mostly same between the two fission yeasts, the DNA damage checkpoint was the only pathway that governed DNA damage-dependent hyphal growth. We also examined whether DNA damage checkpoint signaling engaged in functional crosstalk with other hyphal differentiation pathways because hyphal differentiation can also be triggered by nutritional stress. Here, we discovered genetic interactions that indicated that the cAMP pathway engaged in crosstalk with Chk1-dependent signaling.     

The synthesis of coliphage T1 DNA: Requirement for host dna genes involved in elongation

Available Escherichia coli mutants with temperature-sensitive mutations in genes essential for DNA replication have been used to investigate the host protein involvement in the DNA synthesis of bacteriophage T1. This study has shown that T1 is independent of the E. coli initiation gene products dnaA, dnaC, dnaI, dnaP, and dnaT. The gene products of the polC, dnaG, and dnaZ loci, know to be involved in the elongation events of replication, are required for a productive T1 infection. T1 was found to be independent of the functioning of the dnaB gene product in the four dnaB mutants tested.