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G Mosig

Publications and source records attributed to G Mosig.

At least 19 recordsLinked to original sources
PubMed2001

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer.

Two major pathways of recombination-dependent DNA replication, "join-copy" and "join-cut-copy," can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Amino Acid Sequence↗
PubMed1999

Two new early bacteriophage T4 genes, repEA and repEB, that are important for DNA replication initiated from origin E.

Two new, small, early bacteriophage T4 genes, repEA and repEB, located within the origin E (oriE) region of T4 DNA replication, affect functioning of this origin. An important and unusual property of the oriE region is that it is transcribed at early and late periods after infection, but in opposite directions (from complementary DNA strands). The early transcripts are mRNAs for RepEA and RepEB proteins, and they can serve as primers for leading-strand DNA synthesis. The late transcripts, which are genuine antisense RNAs for the early transcripts, direct synthesis of virion components. Because the T4 genome contains several origins, and because recombination can bypass a primase requirement for retrograde synthesis, neither defects in a single origin nor primase deficiencies are lethal in T4 (Mosig et al., FEMS Microbiol. Rev. 17:83-98, 1995). Therefore, repEA and repEB were expected and found to be important for T4 DNA replication only when activities of other origins were reduced. To investigate the in vivo roles of the two repE genes, we constructed nonsense mutations in each of them and combined them with the motA mutation sip1 that greatly reduces initiation from other origins. As expected, T4 DNA synthesis and progeny production were severely reduced in the double mutants as compared with the single motA mutant, but early transcription of oriE was reduced neither in the motA nor in the repE mutants. Moreover, residual DNA replication and growth of the double mutants were different at different temperatures, suggesting different functions for repEA and repEB. We surmise that the different structures and protein requirements for functioning of the different origins enhance the flexibility of T4 to adapt to varied growth conditions, and we expect that different origins in other organisms with multiorigin chromosomes might differ in structure and function for similar reasons.

Amino Acid Sequence↗
PubMed1998

Several new bacteriophage T4 genes, mapped by sequencing deletion endpoints between genes 56 (dCTPase) and dda (a DNA-dependent ATPase-helicase) modulate transcription.

We have analyzed DNA of wild-type T4 and of 13 independent large viable deletions isolated by Homyk and Weil (Virology 61 (1974) 505-523) and by Little (Virology 53 (1973) 47-59), by sequencing, cloning, and expression studies. The deletions can be explained by illegitimate recombination between short (4- to 15-bp) ectopic repeats. In four deletions, adjacent regions are partially homologous, and in at least one of them, the base adjacent to the overlap was deleted during recombination. The sequence 5'-GGGC, which has not been associated with T4 deletions in other map regions, occurs within three repeats, and near the repeats in four more of the 13 deletions. Five previously named genes, 69, soc, mrh, modA, and dda were mapped relative to the deletion endpoints. Nine additional ORFs were found interspersed between them. One of these shares some similarities with mrh (modulates rpoH; Frazier and Mosig, Gene 88 (1990) 7-14), and another one resembles modA (coding for an ADP-ribosyl-transferase that modifies RNA polymerase alpha subunits, Skórko et al., Eur. J. Biochem. 79 (1977) 55-66) respectively. We found that the host's heat shock sigma factor, sigma32, is phosphorylated, and that Mrh protein modulates this phosphorylation. The ORF dda.9 downstream of mrh has a patchy similarity with conserved C-terminal segments (motifs) of sigma32; therefore, we call it srh. Another ORF, dda.2 located between modA and dda, shares sequence similarity with sigma70, and we call it srd. We consider the possibility that Srh and Srd act as decoys for sigma32, or sigma70, respectively. Expression of several of the ORFs from cloned DNA appears to be toxic to the host bacteria. Mutant clones only could be constructed from gene 69 and from modA. Moreover, dda.2 (srd)-containing bacteria grow extremely slowly, and they form filaments in liquid cultures. Clones carrying mrh and srh show less severe filamentation. Our results highlight the importance of 'non-essential' genes for phage development and evolution.

Amino Acid Sequence↗
PubMed1998

The largest (70 kDa) product of the bacteriophage T4 DNA terminase gene 17 binds to single-stranded DNA segments and digests them towards junctions with double-stranded DNA.

Bacteriophage terminases are oligomeric multifunctional proteins that bind to vegetative DNA, cut it and, together with portal proteins, translocate the DNA into preformed heads. Most terminases are encoded by two partially overlapping genes. In phage T4 they are genes 16 and 17. We have shown before that the larger of these, gene 17, can yield, in addition to a full-length 70 kDa product, several shorter peptides. At least two of these, gene product (gp) 17' and gp17", are initiated in the same reading frame as the 70 kDa gp17 from internal ribosome binding sites. Most of the shorter gp17 s contain predicted ATPase motifs, but only the largest (70 kDa) peptide has a predicted single-stranded DNA binding domain. Here we describe the DNA binding and cutting properties of the purified 70 kDa protein, expressed from two different clones containing gene 17 but no other T4 gene. Epitope-specific antibodies, which recognize several different gene 17 products in extracts of induced clones or of T4-infected cells, precipitate the purified 70 kDa gp17. When Mg2+ is chelated by EDTA this 70 kDa protein binds to single-stranded DNA, preferentially to junctions of single- and double-stranded DNA segments. It does not bind to blunt-ended double-stranded DNA. When Mg2+ is present the purified 70 kDa gp17 digests single-stranded segments preferentially up to junctions with double-stranded DNA. A 70 kDa gp17 from a P379L temperature sensitive (ts) mutant, which has lost the nuclease and ATPase activities, retains the single-stranded DNA binding activity. Taken together with earlier findings these results support a model for packaging of T4 DNA from single-stranded regions in recombinational or replicative intermediates, which occur at nearly random positions of the genome. This mechanism may be an alternative to site-specific initiation of packaging proposed by other investigators.

Adenosine Triphosphatases↗
PubMed1998

A species barrier between bacteriophages T2 and T4: exclusion, join-copy and join-cut-copy recombination and mutagenesis in the dCTPase genes.

Bacteriophage T2 alleles are excluded in crosses between T2 and T4 because of genetic isolation between these two virus species. The severity of exclusion varies in different genes, with gene 56, encoding an essential dCT(D)Pase/dUT(D)Pase of these phages, being most strongly affected. To investigate reasons for such strong exclusion, we have (1) sequenced the T2 gene 56 and an adjacent region, (2) compared the sequence with the corresponding T4 DNA, (3) constructed chimeric phages in which T2 and T4 sequences of this region are recombined, and (4) tested complementation, recombination, and exclusion with gene 56 cloned in a plasmid and in the chimeric phages in Escherichia coli CR63, in which growth of wild-type T2 is not restricted by T4. Our results argue against a role of the dCTPase protein in this exclusion and implicate instead DNA sequence differences as major contributors to the apparent species barrier. This sequence divergence exhibits a remarkable pattern: a major heterologous sequence counter-clockwise from gene 56 (and downstream of the gene 56 transcripts) replaces in T2 DNA the T4 gene 69. Gene 56 base sequences bordering this substituted region are significantly different, whereas sequences of the dam genes, adjacent in the clockwise direction, are similar in T2 and in T4. The gene 56 sequence differences can best be explained by multiple compensating frameshifts and base substitutions, which result in T2 and T4 dCTPases whose amino acid sequences and functions remain similar. Based on these findings we propose a model for the evolution of multiple sequence differences concomitant with the substitution of an adjacent gene by foreign DNA: invasion by the single-stranded segments of foreign DNA, nucleated from a short DNA sequence that was complementary by chance, has triggered recombination-dependent replication by "join-copy" and "join-cut-copy" pathways that are known to operate in the T-even phages and are implicated in other organisms as well. This invasion, accompanied by heteroduplex formation between partially similar sequences, and perhaps subsequent partial heteroduplex repair, simultaneously substituted T4 gene 69 for foreign sequences and scrambled the sequence of the dCTPase gene 56. We suggest that similar mechanisms can mobilize DNA segments for horizontal transfer without necessarily requiring transposase or site-specific recombination functions.

Alleles↗
PubMed1998

Recombination and recombination-dependent DNA replication in bacteriophage T4.

General recombination is essential for growth of phage T4, because origin initiation of DNA replication is inactivated during development, and recombination-dependent initiation is necessary for continuing DNA replication. The requirement of recombination for T4 growth has apparently been a driving force to acquire and maintain multiple recombination mechanisms. This requirement makes this phage an excellent model to analyze several recombination mechanisms that appear redundant under optimal growth conditions but become essential under other conditions, or at different stages of the developmental program. The most important substrate for wild-type T4 recombination is single-stranded DNA generated by incomplete replication of natural or artificial chromosomal ends, or by nucleolytic degradation from induced breaks, or nicks. Recombination circumvents the further erosion of such ends. There are multiple proteins and multiple pathways to initiate formation of recombinants (by single-strand annealing or by strand invasion) and to convert recombinational intermediates into final recombinants ("cut and paste" or "cut and package"), or to initiate extensive DNA replication by "join-copy" or "join-cut-copy" mechanisms. Most T4 recombination is asymmetrical, favoring the initiation of replication. In wild-type T4 these pathways are integrated with physiological changes of other DNA transactions: mainly replication, transcription, and packaging. DNA replication and packaging enzymes participate in recombination, and recombination intermediates supply substrates for replication and packaging. The replicative recombination pathways are also important for transmission of intron DNA to intronless genomes ("homing"), and are implicated in horizontal transfer of foreign genes during evolution of the T-even phages. When horizontal transfer involves heteroduplex formation and repair, it is intrinsically mutagenic and contributes to generation of species barriers between phages.

Bacterial Proteins↗
PubMed1997

A novel mechanism of virus-virus interactions: bacteriophage P2 Tin protein inhibits phage T4 DNA synthesis by poisoning the T4 single-stranded DNA binding protein, gp32.

P2 prophages have been known to inhibit DNA replication and growth of T-even phages. We show here that this inhibition is due to poisoning of the T-even single-stranded DNA binding protein gp32 by the product of the nonessential P2 tin gene. Synthesis of Tin protein from a gene cloned in a multicopy plasmid is necessary and sufficient to completely prevent de novo DNA replication and growth of wild-type T2 or T4 phage. We isolated more than 20 independent mutants that render T-even phages resistant to poisoning by the P2 Tin protein. In all of these mutants, which we call asp, Asp codon 163 of gene 32 is changed to a Gly or Asn codon. The mutant alleles are recessive; i.e., when wild-type and asp mutants coinfect the same host cells, most DNA replication is poisoned by P2 Tin protein. To explain our results, we propose that the P2 Tin protein interacts with T-even gp32 at position 163 and distorts the helical filament of gene 32 protein on single-stranded DNA. Thereby Tin protein inhibits either assembly or function, or both, of the T4 replisome. The inhibition of late gene expression by P2 Tin protein may be an indirect consequence of inhibition of DNA replication.

Bacteriophage P2↗
PubMed1996

Expression of the bacteriophage T4 DNA terminase genes 16 and 17 yields multiple proteins.

The products of the bacteriophage T4 terminase genes 16 and 17 are known to mediate cutting and packaging of concatemeric vegetative DNA. We show here that the larger of these genes, 17, yields multiple protein species. The complex expression of the T4 terminase genes includes overlapping transcripts, probably initiated from multiple promoters, RNA processing at certain preferred sites and translation initiation from multiple ribosome binding sites (RBS). Translation initiation from these RBS may be modulated by inverted repeat (IR) sequences whose folding can be predicted to differ in different RNA species. In T4 infected bacteria, genes 16 and 17 are probably co-transcribed from several near-consensus late promoters upstream from gene 16, and processed at multiple sites. Additional 5' ends of late transcripts are located downstream from a near-consensus late promoter inside gene 17 and further downstream, unrelated to any known promoter consensus sequence. The gene 17 transcripts that are initiated or cleaved internally contain RBS for shorter open reading frames (ORFs) in the same frame as full-length gene product (gp) 17 of 70 kDa. The truncated proteins, a 59-kDa gp17' and a 45-kDa gp17", are synthesized from cloned gene 17 segments in which the first gene 17 RBS is deleted. Expression of gene 17 is different in BL21(DE3) or W3110[pACT7] host bacteria. The gp17' and gp17" proteins are predicted to contain one or more of the ATPase motifs that are common among large subunits of other phage terminases. They lack a predicted single stranded (ss) DNA binding motif that is unique the large terminase proteins in T4 gp17, and that has been implicated in recognizing ssDNA regions in replicating and recombining T4DNA destined to be packaged. We hypothesize that a truncated gene 17' is an evolutionary precursor of the full-size T4 gene 17. Its function may have been maintained to allow processive packaging from double stranded (ds) DNA ends.

Bacteriophage T4↗
PubMed1995

Two copies of a DNA element, 'Wendy', in the chloroplast chromosome of Chlamydomonas reinhardtii between rearranged gene clusters.

We have characterized two copies of a 2.4 kb DNA element that we call 'Wendy', in the chloroplast chromosome of Chlamydomonas reinhardtii. The two copies of Wendy reside in different single-copy regions at opposite positions in the chloroplast genome. Like many mobile DNA elements, both copies of Wendy are bordered by inverted repeats and contain several additional degenerate copies of these repeat sequences in direct or inverted orientation. In addition, four basepairs are repeated in direct orientation. Two major open reading frames (ORFs) are predicted from the DNA sequence of Wendy I. These ORFs are co-transcribed from a promoter inside the element. The deduced amino acid sequence of the larger of these ORFs shares some weak similarities with sequence motifs of transposases and integrases of other mobile elements. Wendy II appears to be altered relative to Wendy I by point mutations and small deletions and insertions which destroy the ORFs. The leader sequence of the Wendy transcript is nearly identical with the leader sequence of the rbcL transcript of C. reinhardtii, but not of C. moewusii (where the complete Wendy was also undetectable). Furthermore, both copies of Wendy are bracketed by gene clusters that are separated in C. reinhardtii but are contiguous in C. moewusii where they exist in an inverted orientation compared with C. reinhardtii. Wendy was not found in any of the completely sequenced chloroplast genomes of rice, tobacco, pine, Euglena or Marchantia, nor in any other GenBank entry. Our results suggest that Wendy has invaded C. reinhardtii after divergence from other species. Subsequent Wendy-dependent illegitimate homologous or site-specific recombination events or both may have contributed to scrambling of the C. reinhardtii chloroplast genome relative to genomes of other species.

Amino Acid Sequence↗
PubMed1995

Multiple initiation mechanisms adapt phage T4 DNA replication to physiological changes during T4's development.

We summarize the evidence for multiple pathways to initiate phage T4 DNA replication. In any infecting chromosome, leading DNA strands can be primed from pre-replicative transcripts, independent of primase activity, at one of several origins. Within each origin region, there are multiple RNA-DNA transition sites. However, the priming potential at each single site is very low. Our results suggest that origin transcripts can become primers for leading strand DNA synthesis without being processed, but that a promoter-proximal segment of each origin transcript plays an important structural role, as a proposed wedge, in the transition from RNA to DNA synthesis. Two recombination-dependent pathways render subsequent phage T4 DNA replication independent of transcription. The first of these requires proteins that are synthesized during the pre-replicative phase of infection. It is active as soon as the first growing points, initiated at origins, have reached a chromosomal end. The other one requires at least one late protein: endonuclease VII, a resolvase that cuts recombinational junctions. The latter pathway can bypass primase deficiencies by allowing retrograde DNA synthesis without Okazaki pieces. We discuss the integration of these multiple and redundant pathways into the developmental program of T4. Competition between these initiation mechanisms and with other DNA transactions allows for integration of replication controls with transcription, recombination and packaging of the DNA.

Bacteriophage T4↗
PubMed1993

Roles of novobiocin-sensitive topoisomerases in chloroplast DNA replication in Chlamydomonas reinhardtii.

We have examined DNA replication in Chlamydomonas reinhardtii chloroplasts in vivo when chloroplast type II topoisomerases are inactivated with sublethal doses of novobiocin. DNA replication is at first inhibited under these conditions. However, after a delay of several hours, chloroplast chromosomes initiate a novobiocin-insensitive mode of DNA replication. This replication starts preferentially near a hotspot of recombination in the large inverted repeats, instead of from the normal chloroplast origins, oriA and oriB. It replicates one, but not the other single-copy region of the chloroplast chromosome. We speculate that novobiocin-insensitive DNA replication in chloroplasts requires recombination in this preferred initiation region.

Animals↗
PubMed1991

Intercalation of psoralen into DNA of plastid chromosomes decreases late during barley chloroplast development.

We have used a DNA crosslinking assay to measure intercalation of the psoralen derivative HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into barley (Hordeum vulgare) plastid chromosomal DNA during chloroplast and etioplast development. Intercalation into DNA in intact plastids in vivo and in plastid lysates in vitro shows that chromosomal DNA in the most mature chloroplasts intercalates HMT less efficiently than DNA in younger chloroplasts. In contrast, there is no change in HMT intercalation during etioplast differentiation in the dark. Our results also show that DNA in higher plant plastid chromosomes is under superhelical tension in vivo. The lower susceptibility to HMT intercalation of DNA in the most mature chloroplasts indicates that late during chloroplast development the superhelical tension or the binding of proteins to the DNA or both change.

Blotting, Southern↗
PubMed1991

Two bacteriophage T4 base plate genes (25 and 26) and the DNA repair gene uvsY belong to spatially and temporally overlapping transcription units.

The bacteriophage T4 DNA recombination-repair gene uvsY located at or near an origin of DNA replication and adjacent to the late base plate genes 25 and 26. Our present results reveal a complex transcription pattern in the region encompassing these genes. Most significantly, uvsY and two ORFs, downstream of it, all of which are transcribed from a middle promoter before the onset of DNA replication, are also part of a larger late transcription unit which includes the base plate genes 25 and 26. The late genes 25 and 26 are transcribed not only late, but also early from one or several early promoters further upstream. Translation, however, is inhibited by secondary structures which sequester the ribosome binding site in the early transcript. We discuss possible advantages of these transcriptional patterns for T4 DNA recombination, replication, and repair. The predicted and in vivo-expressed 23.9-kDa product of gene 26 is smaller than the reported size of gene 26 protein isolated from base plates, suggesting that nascent gp26 might be processed to a larger protein during assembly.

Base Sequence↗
PubMed1991

Bypass of a primase requirement for bacteriophage T4 DNA replication in vivo by a recombination enzyme, endonuclease VII.

A primase, the product of phage T4 gene 61, is required to initiate synthesis of Okazaki pieces and to allow bidirectional replication from several T4 origins. However, primase-defective T4 gene 61 mutants are viable. In these mutants, leading-strand DNA synthesis starts at the same time as in wild type infections, but, in contrast to wild type, initiation is unidirectional and the first replicative intermediates are large displacement loops. Rapid double-strand DNA replication occurs later after infection, generating multiple branched concatemers, which are cut and packaged into viable progeny particles, as in wild-type T4. Evidence is presented that this late double-strand DNA replication requires functional endonuclease VII (endo VII), the product of the T4 gene 49. We propose that endo VII can provide a backup mechanism when primase is defective, because it cuts recombinational junctions, generating 3' ends. These ends can prime DNA synthesis to copy the DNA strands that had been displaced during the initial origin-dependent replication. We explain the DNA-delay phenotype and the commonly observed temperature dependence of DNA replication in primase-deficient gene 61 mutants as a consequence of temperature-dependent translational control of gene 49 expression. In the presence or absence of functional primase endo VII is essential for correct packaging of DNA. The powerful selection that keeps the function of endo VII and expression of its gene at levels that are optimal for T4 development determines both the efficiency and the limitations of the bypass mechanism.

Base Sequence↗
PubMed1990

Light affects the structure of Chlamydomonas chloroplast chromosomes.

We have analyzed changes in the structure of chloroplast chromosomes in response to light in growing Chlamydomonas cells using a crosslinking assay based on the intercalation of HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into DNA. Our results show that the structure of chloroplast chromosomes in at least three widely separated regions is different in light-grown vs. dark-grown cells. Structural changes in chloroplast chromosomes occur within 3 hrs after exposure to light or darkness, respectively. The response to light is not inhibited by atrazine and can be elicited by dim blue light incapable of evolving O2, indicating that it does not require photosynthesis. Inhibition of cytoplasmic protein synthesis with cycloheximide prevents this response to light, indicating that it depends, at least in part, on proteins imported from the cytoplasm.

Atrazine↗
PubMed1990

The bacteriophage T4 gene mrh whose product inhibits late T4 gene expression in an Escherichia coli rpoH (sigma 32) mutant.

In an Escherichia coli rpoH mutant (affecting sigma 32, heat-shock sigma factor) infected at high temperatures with wild-type T4 phage, late T4 transcription and consequently progeny production are dramatically impaired. This defect is due, in part, to insufficient activity of sigma 70 [Frazier and Mosig, J. Bacteriol. 170 (1988) 1384-1388], which is necessary to initiate early T4 transcription. Unexpectedly, however, we found that, in this rpoH host, late T4 transcription is also impaired when the temperature is raised from 30 to 42 degrees C late after infection, when T4 transcription is directed by the T4-encoded sigma factor, sigma gp55. Here, we show that a T4 gene that we call mrh (modulates rpoH), located at 14 kb on the T4 map, is responsible for the inhibition of late T4 transcription in the rpoH mutant host. T4 deletion mutants that lack the mrh gene can produce progeny in the rpoH host, but the Mrh protein, provided in trans from a plasmid-borne mrh gene, inhibits this growth. We have cloned and sequenced this T4 gene and synthesized the Mrh protein in a T7 RNA polymerase-dependent expression system. The Mr of the Mrh protein deduced from the nucleotide sequence is 13419. Gene mrh is cotranscribed with several other, yet unidentified genes, both from an early promoter downstream from the late soc gene (encoding the small outer capsid protein) and from the late soc promoter further upstream.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗