Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase1

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V0) and a peripherally associated catalytic sector (V1). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V0 sector as well as for V1 attachment. Vma6p has been characterized as a nonintegrally associated V0 subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.

Suppression of O-Methyltransferase Gene by Homologous Sense Transgene in Quaking Aspen Causes Red-Brown Wood Phenotypes1

Homologous sense suppression of a gene encoding lignin pathway caffeic acid O-methyltransferase (CAOMT) in the xylem of quaking aspen (Populus tremuloides Michx.) resulted in transgenic plants exhibiting novel phenotypes with either mottled or complete red-brown coloration in their woody stems. These phenotypes appeared in all independent transgenic lines regenerated with a sense CAOMT construct but were absent from all plants produced with antisense CAOMT. The CAOMT sense transgene expression was undetectable, and the endogenous CAOMT transcript levels and enzyme activity were reduced in the xylem of some transgenic lines. In contrast, the sense transgene conferred overexpression of CAOMT and significant CAOMT activity in all of the transgenic plants' leaves and sclerenchyma, where normally the expression of the endogenous CAOMT gene is negligible. Thus, our results support the notion that the occurrence of sense cosuppression depends on the degree of sequence homology and endogene expression. Furthermore, the suppression of CAOMT in the xylem resulted in the incorporation of a higher amount of coniferyl aldehyde residues into the lignin in the wood of the sense plants. Characterization of the lignins isolated from these transgenic plants revealed that a high amount of coniferyl aldehyde is the origin of the red-brown coloration—a phenotype correlated with CAOMT-deficient maize (Zea mays L.) brown-midrib mutants.

Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites

A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.

Characterization of a Novel Human SMC Heterodimer Homologous to the Schizosaccharomyces pombe Rad18/Spr18 Complex

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27–28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

Rescue of arrested replication forks by homologous recombination

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for double-strand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes.

Links between replication and recombination in Saccharomyces cerevisiae: A hypersensitive requirement for homologous recombination in the absence of Rad27 activity

The RAD27 gene of Saccharomyces cerevisiae encodes a 5′-3′ flap exo/endonuclease, which plays an important role during DNA replication for Okazaki fragment maturation. Genetic studies have shown that RAD27 is not essential for growth, although rad27Δ mutants are temperature sensitive. Moreover, they exhibit increased sensitivity to alkylating agents, enhanced spontaneous recombination, and repetitive DNA instability. The conditional lethality conferred by the rad27Δ mutation indicates that other nuclease(s) can compensate for the absence of Rad27. Indeed, biochemical and genetical analyses indicate that Okazaki fragment processing can be assured by other enzymatic activities or by alternative pathways such as homologous recombination. Here we present the results of a screen that makes use of a synthetic lethality assay to identify functions required for the survival of rad27Δ strains. Altogether, we confirm that all genes of the Rad52 recombinational repair pathway are required for the survival of rad27Δ strains at both permissive (23°C) and semipermissive (30°C) temperatures for growth. We also find that several point mutations that confer weaker phenotypes in mitotic than in meiotic cells (rad50S, mre11s) and additional gene deletions (com1/sae2, srs2) exhibit synthetic lethality with rad27Δ and that rad59Δ exhibits synergistic effects with rad27Δ. This and previous studies indicate that homologous recombination is the primary, but not only, pathway that functions to bypass the replication defects that arise in the absence of the Rad27 protein.

Repeat expansion by homologous recombination in the mouse germ line at palindromic sequences

Genetic instability can be induced by unusual DNA structures and sequence repeats. We have previously demonstrated that a large palindrome in the mouse germ line derived from transgene integration is extremely unstable and undergoes stabilizing rearrangements at high frequency, often through deletions that produce asymmetry. We have now characterized other palindrome rearrangements that arise from complex homologous recombination events. The structure of the recombinants is consistent with homologous recombination occurring by a noncrossover gene conversion mechanism in which a break induced in the palindrome promotes homologous strand invasion and repair synthesis, similar to mitotic break repair events reported in mammalian cells. Some of the homologous recombination events led to expansion in the size of the palindromic locus, which in the extreme case more than doubled the number of repeats. These results may have implications for instability observed at naturally occurring palindromic or quasipalindromic sequences.

Homologous DNA recombination in vertebrate cells

The RAD52 epistasis group genes are involved in homologous DNA recombination, and their primary structures are conserved from yeast to humans. Although biochemical studies have suggested that the fundamental mechanism of homologous DNA recombination is conserved from yeast to mammals, recent studies of vertebrate cells deficient in genes of the RAD52 epistasis group reveal that the role of each protein is not necessarily the same as that of the corresponding yeast gene product. This review addresses the roles and mechanisms of homologous recombination-mediated repair with a special emphasis on differences between yeast and vertebrate cells.

Manipulating the mammalian genome by homologous recombination

Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies—chimeric nucleases and triplex-forming oligonucleotides—for stimulating recombination in cells.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Two highly homologous ribonuclease genes expressed in mouse eosinophils identify a larger subgroup of the mammalian ribonuclease superfamily.

Two putative ribonucleases have been isolated from the secondary granules of mouse eosinophils. Degenerate oligonucleotide primers inferred from peptide sequence data were used in reverse transcriptase-PCR reactions of bone marrow-derived cDNA. The resulting PCR product was used to screen a C57BL/6J bone marrow cDNA library, and comparisons of representative clones showed that these genes and encoded proteins are highly homologous (96% identity at the nucleotide level; 92/94% identical/similar at the amino acid level). The mouse proteins are only weakly homologous (approximately 50% amino acid identity) with the human eosinophil-associated ribonucleases (i.e., eosinophil-derived neurotoxin and eosinophil cationic protein) and show no sequence bias toward either human protein. Phylogenetic analyses established that the human and mouse loci shared an ancestral gene, but that independent duplication events have occurred since the divergence of primates and rodents. The duplication event generating the mouse genes was estimated to have occurred < 5 x 10(6) years ago (versus 30 to 40 x 10(6) years ago in primates). The identification of independent duplication events in two extant mammalian orders suggests a selective advantage to having multiple eosinophil granule ribonucleases. Southern blot analyses in the mouse demonstrated the existence of three additional highly homologous genes (i.e., five genes total) as well as several more divergent family members. The potential significance of this observation is the implication of a larger gene subfamily in primates (i.e., humans).

A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange.

Activation of the ubiquitously expressed Na-H exchanger, NHE1, results in an increased efflux of intracellular H+. The increase in intracellular pH associated with this H+ efflux may contribute to regulating cell proliferation, differentiation, and neoplastic transformation. Although NHE1 activity is stimulated by growth factors and hormones acting through multiple GTPase-mediated pathways, little is known about how the exchanger is directly regulated. Using expression library screening, we identified a novel protein that specifically binds to NHE1 at a site that is critical for growth factor stimulation of exchange activity. This protein is homologous to calcineurin B and calmodulin and is designated CHP for calcineurin B homologous protein. Like NHE1, CHP is widely expressed in human tissues. Transient overexpression of CHP inhibits serum- and GTP-ase-stimulated NHE1 activity. CHP is a phosphoprotein and expression of constitutively activated GTPases decreases CHP phosphorylation. The phosphorylation state of CHP may therefore be an important signal controlling mitogenic regulation of NHE1.