Top PDF Host- and Phage-Mediated Repair of Radiation Damage in Bacteriophage T4

Escherichia coli str-ains Desi-nation Genotype with respect to repair B Wild B.,-, H/r30R uCvr-e vr-e H s30R uvrA res* R15 N1071 N1072 N1 252 Source Wild UV", X-ray sensitive Radioresist[r]

An amber mutation in gene 49 decreased recombina- Our results indicate that repair from the topo site tional repair about fourfold with either the topo site or requires the same recombinational repair proteins as the I-TevI site, suggesting a role for this protein in the repair of endonuclease-generated DSBs. Both types of repair mechanism. The residual repair in the gene 49 repair are largely dependent on the UvsX and UvsY mutant might reflect repair events that do not require proteins and absolutely require gp46 and gp32. In addi- the Holliday junction resolvase. Alternatively, some tion, both types of repair are reduced severalfold by other protein(s) might resolve Holliday junctions in the an amber mutation in gene 49. Taken together, these absence of gp49, or a small amount of gp49 might be results strongly suggest that the drug-stabilized cleavage expressed despite the amber mutation. complexes at the topo site are processed into protein- The major conclusion is that repair of both topoisom- free DSBs that are subsequently acted upon by the phage erase-mediated damage and I-TevI breaks requires the recombinational repair apparatus. Nonetheless, two re- same proteins, which argues for very similar repair sults indicate that only a small fraction of detectable

KOZINSKI of Human Genetics, University of Pennsylvania School of Medicine, Philadelphia, Department Pennsylvania 19104 Discontinuities of T4 DNA which are caused by excision of UV-damage[r]

by infection with wild-type or Dsrd mutant. RNAs extracted from cells at appropriate times after infection were analyzed by Northern blotting. Full-length lpp mRNA was stabilized, and the decay intermediate was hardly detected in RNase E-defective cells infected with Dsrd mutant as well as wild- type phage (Figure 3, A and B). The cleavage site to generate this decay intermediate was determined by primer extension analysis, and its 59-terminus was two nucleotides down- stream of the start codon of lpp mRNA (AUGAYAA) (Figure 3C and Figure S3). RNase E has no canonical target sequence for cleavage, but preferentially cleaves at regions that are single-stranded with AU-rich sequences. These facts clearly support the idea that RNase E generates this decay interme- diate. Finally, we examined the effect of RppH on decay of lpp and ompA mRNAs stimulated by Srd (Figure 3, D and E). After infection with wild-type phage, both mRNAs were de- graded in DrppH cells as fast as in wild-type cells, indicating

lesions, and, in the case of smc6-74, a further defect in the maintenance of the Chk1-dependent checkpoint arrest exists, despite normal activation of Chk1 activity (V erkade and O’C onnell 1998; V erkade et al. 1999; H arvey et al. 2004). Spore germination experiments with cells deleted for either smc6 or nse1 and the analysis of additional smc6 alleles have confirmed that this complex is indeed required to successfully respond to DNA damage and pass through mitosis. Further, condi- tional and hypomorphic alleles of nse1, nse2, nse3, and nse4, with a conditional allele of rad60, which encodes a protein involved in Smc5/6 function without being a member of the complex per se (M orishita et al. 2002; B oddy et al. 2003), also show defects in DNA damage responses. Recent data from S. cerevisiae have demon- strated a defect in the segregation of rDNA at mitosis in temperature-sensitive smc5-6 and smc6-9 mutants, lead- ing to DNA damage that can be partially suppressed in rad52 mutants, suggesting inappropriate recombina- tion at the repetitive rDNA (T orres -R osell et al. 2005). This may be related to observed epistasis between var- ious smc6 and nse1-nse4 alleles and deletion of the Rad51 homolog, rhp51 in S. pombe (L ehmann et al. 1995; M c D onald et al. 2003; M orikawa et al. 2004; P ebernard et al. 2004). Similar epistasis has been seen between smc6-56 and rad52 D in S. cerevisiae (T orres - R osell et al. 2005), and here the smc6 mutation also results in defects in methyl methanesulfonate (MMS)- induced interchromosomal and sister chromatid re- combination (O noda et al. 2004). Presumably, as with condensin and cohesin, the defective DNA damage responses of mutants in the Smc5/6 complex are a con- sequence of a more fundamental defect in chromosome organization.

the cleaved plasmid DNA, which was being produced earlier than normal in the phage infection. E. coli exo- nuclease V (RecBCD) is known to be highly active on double-stranded ends and is thought to be active during the early part of T4 infections (Lipinska et al. 1989; Appasani et al. 1999). We moved plasmid pTD001 into a host strain that carried the recB21 mutation, which inactivates exonuclease V, and repeated the assay with phage ITM. Under these conditions, cleaved plasmid DNA accumulated to high levels and both types of dele- tion product were evident (Figure 4A, lane 5). Infection with the double mutant ITM-46 ⫺ resulted in a large accumulation of cleaved plasmid DNA, as well as the production of a significant amount of unreplicated dele- tion product at late times (Figure 4A, lane 6). For both the 46 ⫹ and 46 ⫺ phage, the amount of cleaved plasmid DNA was increased relative to comparable infections in Figure 4.—Altered timing of I-TevI expression and impact the recB ⫹ host (Figure 4A, compare lanes 5 and 6 to 2 of recB mutation on DSBR. Plasmid DNA from infected cells and 3). We conclude that E. coli exonuclease V activity was digested with AflIII and analyzed by Southern blotting

chloramphenicol with light 32P-labeled UVThis observation made it necessary to ascertain irradiated rII-vi phage A, the same UVwhether sufficient quantities of functional v irradiated ph[r]

COLI rial as well as other unknown cell components from uninfected cells; ii the increased rate of release of envelope material and other substances rapidly upon phage infection; iii the[r]

The variation i n the frequency with which different rll-diploids produce r- segregants and in the ratio with which the two types of r-segregants are produced indicates that[r]

When 100 phages per sphaeroplast are added to the system, a decreased number of infective centres of the recipient rZZ 1272 phage is observed relative to the introduced phage number[r]

of T4 had been identified. In 1960, when Dick Epstein and I started to work When I received the invitation to write this Perspectives with conditional-lethal mutants, our knowledge of the article, I had almost nothing at hand except my memo- genome of T4 was quite sketchy. Until 1960 we had ries. I could not find any of my reprints, and my science been living with the view that T4 had three linkage files had long since disappeared. I retired in 1990, and groups, perhaps three or more DNA molecules, which for the last 14 years I have engaged almost entirely in duplicated, recombined, and somehow assorted them- nonscientific matters. I recall with some embarrassment selves properly into phage heads at maturation. Figure that 20 or so years ago the Genetics Society of America 1 is the “historical” T4 linkage map I drew for our grant asked that I give my papers to the University Library for report in 1961. It consisted basically of the three linkage

For anaerobic growth, the deaerated medium, usually 792 Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest Cadaverine was found in bacteriophage T4 when the host cells of [r]

The results can be explained, however, if it is lack of agreement as to the effect of inhibiting protein synthesis on the phage's ability to shut hypothesized that phage coats cause a sp[r]

6, 1970 INHIBITION BY GHOSTS by chloramphenicol 23 or amino acid starvation 25, T-even phage infection inhibits host nucleic acid synthesis only partially and to an extent that is multip[r]

If no new ribosomes attached to host mRNA in the T4-infected culture, then no new polysomes would develop and polysomes existent at the time of infection would either stay the same size [r]

Because phage DNA synthesis obscures the degradation pattern of host DNA in cells infected with wild-type T4, these studies were conducted with the double mutant nd28 X amN82 in addition[r]

POLLARD Department of Biophysics, Penntsylvaniia State Uniiversity, Uniiversity Park, Penlntsylvania 16802 Received for publication 24 November 1971 Since the pioneer experiments of Ande[r]

First, the dsb induces a repair reaction that is directly coupled to extensive plasmid replication; the repaired/replicated product is in the form of long plasmid concatemers.. Seco[r]

Our conclusion is based on the following results: i T4amC5 gene 59 is sensitive to both UV irradiation and MMS treatment; ii the results of alkaline sucrose gradient sedimentation of int[r]

This DNA was released from the membrane later in infection as the result of formation of the phage head; detachment of the DNA required the action of gene products 20, 21, 22, 23, 24, 31[r]