Involvement of Gene 49 in Recombination of Bacteriophage T4

The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not.     

Deficiency of double mutant recombinants in crosses of phage T4

Summary Only 1.4% of the double mutant recombinants expected on the basis of wild-type recombination frequencies were observed in the combined data from two-factor crosses between a gene 37 amber mutant, amB280, and eighteen different temperature sensitive mutants which were also defective in gene 37. Similar, though less extreme, deficiencies of double mutant recombinants were observed by Doermann and Parma (1968) for mutants in several other genes. In our amB280xts crosses, frequencies of wild-type recombinants were in reasonably good agreement with those expected from the map positions of the mutants determined in crosses not involving amB280. Wild-type and double mutant recombinants were found at comparable frequencies when each of three other gene 37 amber mutants was crossed to a gene 37 temperature sensitive mutant.Experiments were performed to test whether the deficiency of double mutant recombinants in the amB280xts crosses could be explained by assuming that they occurred primarily in heterozygous particles, where their expression was masked. However, no evidence in support of this explanation was found. Other possible explanations, that the deficiency of double mutants was due to their inviability or the inability of double mutant chromosomes to replicate, were also inconsistent with our observations. The hypothesis considered to most plausibly explain our evidence is that the process by which double mutant recombinant chromosomes are formed is inhibited in the vicinity of a poorly suppressed am mutation.     

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.     

Ultraviolet light sensitive mutants of Coprinus lagopus. I. Isolation and characterization

4UV-sensitive mutants have been isolated from the wild type strain BC9/66 of Coprinus lagopus by following a new replica plating technique. Complementation and recombination studies between these mutants suggest 3 gene loci uvs1, uvs2 and uvs3, two of the mutants being allelic (uvs3-1 and uvs3-2). The mutants uvs2, uvs3-1 and uvs3-2 show photoreactivation whereas the mutant uvs1 appears to be deficient in this respect. None of the mutants of the wild type showed significant recovery after dark holding.     

Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae

In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1Δ mutants are repair-proficient, yen1Δ mus81Δ double mutants are exquisitely sensitive to a variety of DNA-damaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1Δ mus81Δ double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage.     

Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products.

Wild-type bacteriophage T4 and DNA-delay am mutants defective in genes 39, 52, 60 and 58–61 were tested for intracellular sensitivity to the antibiotics coumermycin and novobiocin, drugs which inhibit the DNA gyrase of Escherichia coli. Treatment with these antibiotics drastically reduced the characteristic growth of gene 39, 52 and 60 DNA-delay am mutants in E. coli lacking an amber suppressor (su^?). Wild-type phage-infected cells were unaffected by the drugs while the burst size of a gene 58–61 mutant was affected to an intermediate extent. A su^?E. coli strain which is resistant to coumermycin due to an altered gyrase permitted growth of the DNA-delay am mutants in the presence of the drug. Thus, the characteristic growth of the DNA-delay am mutants in an su^? host apparently depends on the host gyrase. An E. coli himB mutant is defective in the coumermycin-sensitive subunit of gyrase (H. I. Miller, personal communication). Growth of the gene 39, 52 and 60 am mutants was inhibited in the himB mutant while the gene 58–61 mutant and wild-type T4 showed small reductions in burst size in this host. Experiments with nalidixic acid-sensitive and resistant strains of E. coli show that wild-type phage T4 requires a functional nalA protein for growth.Novobiocin and coumermycin inhibit phage DNA synthesis in DNA-delay mutant-infected su^?E. coli if added during the early logarithmic phase of phage DNA synthesis. The gene 58–61 mutant showed the smallest inhibition of DNA synthesis in the presence of the drugs. Addition of the drugs during the late linear phase of phage DNA synthesis had no effect on further synthesis in DNA-delay mutant-infected cells. Coumermycin and novobiocin had no effect on DNA synthesis in wild-type-infected cells regardless of the time of addition of the antibiotics. Models are considered in which the DNA-delay gene products either form an autonomous phage gyrase or interact with the host gyrase and adapt it for proper initiation of phage DNA replication.     

Role of Blm and collaborating factors in recombination and survival following replication stress in Ustilago maydis

Inactivation of the structural gene for the RecQ family member, BLM in human, Sgs1 in budding yeast, or Rqh1 in fission yeast leads to inappropriate recombination, chromosome abnormalities, and disturbed replication fork progression. Studies with yeasts have demonstrated that auxiliary gene functions can contribute in overlapping ways with Sgs1 or Rqh1 to circumvent or overcome lesions in DNA caused by certain genotoxic agents. In the combined absence of these functions, recombination-mediated processes lead to severe loss of fitness. Here we performed a genetic study to determine the role of the Ustilago maydis Blm homolog in DNA repair and in alleviating replication stress. We characterized the single mutant as well as double mutants additionally deleted of genes encoding Srs2, Fbh1, Mus81, or Exo1. Unlike yeasts, neither the blm srs2, blm exo1, nor blm mus81 double mutant exhibited extreme loss of fitness. Inactivation of Brh2, the BRCA2 homolog, suppressed toxicity to hydroxyurea caused by loss of Blm function. However, differential suppression by Brh2 derivatives lacking the canonical DNA-binding region suggests that the particular domain structure comprising this DNA-binding region may be instrumental in promoting the observed hydroxyurea toxicity.     

The role of AtMUS81 in DNA repair and its genetic interaction with the helicase AtRecQ4A

The endonuclease MUS81 has been shown in a variety of organisms to be involved in DNA repair in mitotic and meiotic cells. Homologues of the MUS81 gene exist in the genomes of all eukaryotes, pointing to a conserved role of the protein. However, the biological role of MUS81 varies between different eukaryotes. For example, while loss of the gene results in strongly impaired fertility in Saccharomyces cerevisiae and nearly complete sterility in Schizosaccharomyces pombe, it is not essential for meiosis in mammals. We identified a functional homologue (AtMUS81/At4g30870) in the genome of Arabidopsis thaliana and isolated a full-length cDNA of this gene. Analysing two independent T-DNA insertion lines of AtMUS81, we found that they are sensitive to the mutagens MMS and MMC. Both mutants have a deficiency in homologous recombination in somatic cells but only after induction by genotoxic stress. In contrast to yeast, no meiotic defect of AtMUS81 mutants was detectable and the mutants are viable. Crosses with a hyperrecombinogenic mutant of the AtRecQ4A helicase resulted in synthetic lethality in the double mutant. Thus, the nuclease AtMUS81 and the helicase AtRecQ4A seem to be involved in two alternative pathways of resolution of replicative DNA structures in somatic cells.     

A network of multi-tasking proteins at the DNA replication fork preserves genome stability

To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions) whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics.     

New Late Gene, dar, Involved in DNA Replication of Bacteriophage T4 I. Isolation, Characterization, and Genetic Location

Suppressors of gene 59-defective mutants were isolated by screening spontaneous, temperature-sensitive (ts) revertants of the amber mutant, amC5, in gene 59. Six ts revertants were isolated. No gene 59-defective ts recombinant was obtained by crossing each ts revertant with the wild type, T4D. However, suppressors of gene 59-defective mutants were obtained from two of these ts revertants. These suppressor mutants are referred to as dar (DNA arrested restoration). dar mutants specifically restored the abnormalities, both in DNA synthesis and burst size, caused by gene 59-defective mutants to normal levels. It is unlikely that dar mutants are nonsense suppressors since theý failed to suppress amber mutations in 11 other genes investigated. The genetic expression of dar is controlled by gene 55; therefore, dar is a late gene. The genetic location of dar has been mapped between genes 24 and 25, a region contiguous to late genes. dar appears to be another nonessential gene of T4 since burst sizes of dar were almost identical to those of the wild type. Mutations in dar did not affect genetic recombination and repair of UV-damaged DNA, but caused a sensitivity to hydroxyurea in progeny formation. The effect of the dar mutation on host DNA degradation cannot account for its hydroxyurea sensitivity. dar mutant alleles were recessive to the wild-type allele as judged by restoration of arrested DNA synthesis. The possible mechanisms for the suppression of defects in gene 59 are discussed.     

Rad51-Dependent Aberrant Chromosome Structures at Telomeres and Ribosomal DNA Activate the Spindle Assembly Checkpoint

The spindle assembly checkpoint (SAC) monitors defects in kinetochore-microtubule attachment or lack of tension at kinetochores and arrests cells at prometaphase. In fission yeast, the double mutant between pot1Δ and the helicase-dead point mutant of the RecQ helicase Rqh1 gene (rqh1-hd) accumulates Rad51-dependent recombination intermediates at telomeres and enters mitosis with those intermediates. Here, we found that SAC-dependent prometaphase arrest occurred more frequently in pot1Δ rqh1-hd double mutants than in rqh1-hd single mutants. SAC-dependent prometaphase arrest also occurred more frequently in rqh1-hd single mutants after cells were released from DNA replication block compared to the rqh1-hd single mutant in the absence of exogenous insult to the DNA. In both cases, Mad2 foci persisted longer than usual at kinetochores, suggesting a defect in kinetochore-microtubule attachment. In pot1Δ rqh1-hd double mutants and rqh1-hd single mutants released from DNA replication block, SAC-dependent prometaphase arrest was suppressed by the removal of the recombination or replication intermediates. Our results indicate that the accumulation of recombination or replication intermediates induces SAC-dependent prometaphase arrest, possibly by affecting kinetochore-microtubule attachment.     

γ-Ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telagiectasia cell lines

Well characterized γ-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telagiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characterteristics of γ-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Norman human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline gradient centrifugation, repression of DNA synthesis by low doses of γ-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.     

Nonsense mutants in the bacteriophage T4D v gene

Ten UV-sensitive mutants of T4D with the v phenotype were isolated. Of these ten mutants, two are amber and two opal. In UV curves and in photoreactivation and multiplicity reactivation (MR) experiments the nonsense mutants show the v phenotype in su⁻ hosts and almost the T4+ phenotype in su⁺ hosts. The mutations are located between rI and e and are alleles of v₁. In crosses with irradiated and non-irradiated phages the recombinant frequency is not reduced by uvs5.Amber uvs5 propagated in CR63 su⁺ is with B su⁻ just as sensitive to UV as uvs5 propagated in B su⁻, which permits the conclusion that the capsid of T4 phage particles does not contain the v gene product.In addition, four mutants with a relative UV sensitivity equal to that of T4x were isolated. These are discussed in the next paper³³.     

dRecQ4 Is Required for DNA Synthesis and Essential for Cell Proliferation in Drosophila

Background

The family of RecQ DNA helicases plays an important role in the maintenance of genomic integrity. Mutations in three of the five known RecQ family members in humans, BLM, WRN and RecQ4, lead to disorders that are characterized by predisposition to cancer and premature aging.

Methodology/Principal Findings

To address the in vivo functions of Drosophila RecQ4 (dRecQ4), we generated mutant alleles of dRecQ4 using the targeted gene knock-out technique. Our data show that dRecQ4 mutants are homozygous lethal with defects in DNA replication, cell cycle progression and cell proliferation. Two sets of experiments suggest that dRecQ4 also plays a role in DNA double strand break repair. First, mutant animals exhibit sensitivity to gamma irradiation. Second, the efficiency of DsRed reconstitution via single strand annealing repair is significantly reduced in the dRecQ4 mutant animals. Rescue experiments further show that both the N-terminal domain and the helicase domain are essential to dRecQ4 function in vivo. The N-terminal domain is sufficient for the DNA repair function of dRecQ4.

Conclusions/Significance

Together, our results show that dRecQ4 is an essential gene that plays an important role in not only DNA replication but also DNA repair and cell cycle progression in vivo.     

Dot1 and Set2 histone methylases control the spontaneous and UV-induced mutagenesis levels in the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeasts

In the Saccharomyces cerevisiae yeasts, the DOT1 gene product provides methylation of lysine 79 (K79) of histone H3 and the SET2 gene product provides the methylation of lysine 36 (K36) of the same histone. We determined that the dot1 and set2 mutants suppress the UV-induced mutagenesis to an equally high degree. The dot1 mutation demonstrated statistically higher sensitivity to the low doses of MMC than the wild type strain. The analysis of the interaction between the dot1 and rad52 mutations revealed a considerable level of spontaneous cell death in the double dot1 rad52 mutant. We observed strong suppression of the gamma-induced mutagenesis in the set2 mutant. We determined that the dot1 and set2 mutations decrease the spontaneous mutagenesis rate in both single and double mutants. The epistatic interaction between the dot1 and set2 mutations and almost similar sensitivity of the corresponding mutants to the different types of DNA damage allow one to conclude that both genes are involved in the control of the same DNA repair pathways, the homologous-recombination-based and the postreplicative DNA repair.     

The DNA translocase RAD5A acts independently of the other main DNA repair pathways,and requires both its ATPase and RING domain for activity in Arabidopsis thaliana

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error‐free branch of post‐replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication‐associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single‐strand break repair (AtPARP1), as well as microhomology‐mediated double‐strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM‐mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects.     

Genetic Perturbation of the Maize Methylome

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.