Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

ProDom: automated clustering of homologous domains

The ProDom database is a comprehensive set of protein domain families automatically generated from the SWISS-PROT and TrEMBL sequence databases. An associated database, ProDom-CG, has been derived as a restriction of ProDom to completely sequenced genomes. The ProDom construction method is based on iterative PSI-BLAST searches and multiple alignments are generated for each domain family. The ProDom web server provides the user with a set of tools to visualise multiple alignments, phylogenetic trees and domain architectures of proteins, as well as a BLAST-based server to analyse new sequences for homologous domains. The comprehensive nature of ProDom makes it particularly useful to help sustain the growth of InterPro.     

Therefore,what are recombination proteins there for?

The order of discovery can have a profound effect upon the way in which we think about the function of a gene. In E. coli, recA is nearly essential for cell survival in the presence of DNA damage. However, recA was originally identified, as a gene required to obtain recombinant DNA molecules in conjugating bacteria. As a result, it has been frequently assumed that recA promotes the survival of bacteria containing DNA damage by recombination in which DNA strand exchanges occur. We now know that several of the processes that interact with or are controlled by recA, such as excision repair and translesion synthesis, operate to ensure that DNA replication occurs processively without strand exchanges. Yet the view persists in the literature that recA functions primarily to promote recombination during DNA repair. With the benefit of hindsight and more than three decades of additional research, we reexamine some of the classical experiments that established the concept of DNA repair by recombination, and we consider the possibilities that recombination is not an efficient mechanism for rescuing damaged cells, and that recA may be important for maintaining processive replication in a manner that does not generally promote recombination.     

A new fourier transform approach for protein coding measure based on the format of the Z curve

MOTIVATION: At the core of most protein gene-finding algorithms are the coding measures used to make a decision on coding/non-coding. Of the protein coding measures, the Fourier measure is one of the most important. However, due to the limited length of the windows usually used, the accuracy of the measure is not satisfactory. This paper is devoted to improving the accuracy by lengthening the sequence to amplify the periodicity of 3 in the coding regions. RESULTS: A new algorithm is presented called the lengthen-shuffle Fourier transform algorithm. For the same window length, the percentage accuracy of the new algorithm is 6-7% higher than that of the ordinary Fourier transform algorithm. The resulting percentage accuracy (average of specificity and sensitivity) of the new measure is 84.9% for the window length 162 bp. AVAILABILITY: The program is available on request fromC.- T. Zhang. Contact: ctzhang@tju.edu.cn      

ATP Binding, ATP Hydrolysis, and Protein Dimerization Are Required for RecF to Catalyze an Early Step in the Processing and Recovery of Replication Forks Disrupted by DNA Damage

In Escherichia coli, the recovery of replication following disruption by UV-induced DNA damage requires the RecF protein and occurs through a process that involves stabilization of replication fork DNA, resection of nascent DNA to allow the offending lesion to be repaired, and reestablishment of a productive replisome on the DNA. RecF forms a homodimer and contains an ATP binding cassette ATPase domain that is conserved among eukaryotic SMC (structural maintenance of chromosome) proteins, including cohesin, condensin, and Rad50. Here, we investigated the functions of RecF dimerization, ATP binding, and ATP hydrolysis in the progressive steps involved in recovering DNA synthesis following disruption by DNA damage. RecF point mutations with altered biochemical properties were constructed in the chromosome. We observed that protein dimerization, ATP binding, and ATP hydrolysis were essential for maintaining and processing the arrested replication fork, as well as for restoring DNA synthesis. In contrast, stabilization of the RecF protein dimer partially protected the DNA at the arrested fork from degradation, although overall processing and recovery remained severely impaired.     

BRCA1-directed,enhanced and aberrant homologous recombination: Mechanism and potential treatment strategies

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitin-ligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.Key words: BRCT, DNA repair, peptide, radiation, RING, ubiquitylation     

Terrigenous sediment-dominated reef platform infilling: an unexpected precursor to reef island formation and a test of the reef platform size–island age model in the Pacific

Low-lying coral reef islands are considered highly vulnerable to climate change, necessitating an improved understanding of when and why they form, and how the timing of formation varies within and among regions. Several testable models have been proposed that explain inter-regional variability as a function of sea-level history and, more recently, a reef platform size model has been proposed from the Maldives (central Indian Ocean) to explain intra-regional (intra-atoll) variability. Here we present chronostratigraphic data from Pipon Island, northern Great Barrier Reef (GBR), enabling us to test the applicability of existing regional island evolution models, and the platform size control hypothesis in a Pacific context. We show that reef platform infilling occurred rapidly (~4–5 mm yr⁻¹) under a “bucket-fill” type scenario. Unusually, this infilling was dominated by terrigenous sedimentation, with platform filling and subsequent reef flat formation complete by ~5000 calibrated years BP (cal BP). Reef flat exposure as sea levels slowly fell post highstand facilitated a shift towards intertidal and subaerial-dominated sedimentation. Our data suggest, however, a lag of ~1500 yr before island initiation (at ~3200 cal BP), i.e. later than that reported from smaller and more evolutionarily mature reef platforms in the region. Our data thus support: (1) the hypothesis that platform size acts to influence the timing of platform filling and subsequent island development at intra-regional scales; and (2) the hypothesis that the low wooded islands of the northern GBR conform to a model of island formation above an elevated reef flat under falling sea levels.

     

Nucleotide Excision Repair Is a Predominant Mechanism for Processing Nitrofurazone-Induced DNA Damage in Escherichia coli

Nitrofurazone is reduced by cellular nitroreductases to form N²-deoxyguanine (N²-dG) adducts that are associated with mutagenesis and lethality. Much attention recently has been given to the role that the highly conserved polymerase IV (Pol IV) family of polymerases plays in tolerating adducts induced by nitrofurazone and other N²-dG-generating agents, yet little is known about how nitrofurazone-induced DNA damage is processed by the cell. In this study, we characterized the genetic repair pathways that contribute to survival and mutagenesis in Escherichia coli cultures grown in the presence of nitrofurazone. We find that nucleotide excision repair is a primary mechanism for processing damage induced by nitrofurazone. The contribution of translesion synthesis to survival was minor compared to that of nucleotide excision repair and depended upon Pol IV. In addition, survival also depended on both the RecF and RecBCD pathways. We also found that nitrofurazone acts as a direct inhibitor of DNA replication at higher concentrations. We show that the direct inhibition of replication by nitrofurazone occurs independently of DNA damage and is reversible once the nitrofurazone is removed. Previous studies that reported nucleotide excision repair mutants that were fully resistant to nitrofurazone used high concentrations of the drug (200 μM) and short exposure times. We demonstrate here that these conditions inhibit replication but are insufficient in duration to induce significant levels of DNA damage.Replication in the presence of DNA damage is thought to produce most of the mutagenesis, genomic rearrangements, and lethality that occur in all cells. UV-induced photoproducts, X-ray-induced strand breaks, psoralen- or cis-platin-interstrand cross-links, oxidized bases from reactive oxygen species, and base depurination are just a few of the structurally distinct challenges that the replication machinery must overcome. It seems likely that the mechanisms that process these lesions will vary depending on the nature of the impediment.While a number of the lesions described above are known to block replication, the events associated with UV-induced damage have been the most extensively characterized. UV irradiation causes the formation of cyclobutane pyrimidine dimers and 6-4 photoproducts in DNA that block the progression of the replication fork (16, 29, 30, 37). Following the arrest of replication at UV-induced damage, RecA and several RecF pathway proteins are required to process the replication fork such that the blocking lesion is removed or bypassed (2, 5, 6, 8-10). Cells lacking either RecA or any of several RecF pathway proteins are hypersensitive to UV-induced damage and fail to recover replication following disruption by the lesions (2, 6, 10). RecBCD is an exonuclease/helicase complex that is involved in repairing double-strand breaks (38). It also is required for resistance to UV-induced damage, although it is not required to process or restore disrupted replication forks, and the substrates it acts upon after UV irradiation currently remain unclear (3, 10, 19).Survival and the ability to resume DNA synthesis following UV-induced damage depend predominantly on the removal of the lesions by nucleotide excision repair (5, 7, 36). Cells deficient in nucleotide excision repair are unable to remove UV-induced DNA lesions and exhibit elevated levels of mutagenesis, strand exchanges, rearrangements, and cell lethality (16, 33, 34). In cases where replication fork processing or lesion repair is prevented, the recovery of replication and survival become entirely dependent on translesion synthesis by DNA polymerase V (Pol V) (6). However, in repair-proficient cells, the contribution of translesion synthesis to recovery and survival is minor and is detected only following UV doses that exceed the repair capacity of the cell (5, 6).Less is known about how replication recovers from other forms of DNA damage. We chose to characterize nitrofurazone, because a number of studies suggested that N²-deoxyguanine (N²-dG) adducts induced by this and other agents would be processed differently than UV-induced lesions. Nitrofurazone is a topical antibacterial agent that historically has been used for treating burns and skin grafts in patients and animals (14, 15, 32). Nitrofurazone toxicity is known to require activation by cellular nitroreductases (25, 42). However, the mechanism and targets of its antimicrobial properties have yet to be fully elucidated. In addition to its antimicrobial properties, the reduced nitrofurazone metabolites also target DNA and have been shown to induce free radical damage, strand breaks, and N²-dG adducts (26, 40, 42, 45), and they are mutagenic and carcinogenic in rodent models (1, 15, 24, 39).Whereas nucleotide excision repair is the predominant mechanism required for survival after UV-induced damage, a number of studies suggest that translesion synthesis plays a larger role in survival after nitrofurazone-induced DNA damage. dinB mutants lacking Pol IV were shown to be hypersensitive to nitrofurazone compared to cells that constitutively express the polymerase (17). Biochemically, Pol IV and a number of Pol IV homologs from other organisms have been shown to efficiently replicate over a range of N²-dG adducts in vitro (17, 35, 44). In addition, several studies have reported that uvrA mutants, which are defective in nucleotide excision repair, do not exhibit any hypersensitivity to nitrofurazone or other agents that induce similar adducts in vivo (12, 21, 27). Early studies also observed a direct correlation between nitrofurazone-induced mutations and lethality, suggesting that mutagenic lesions persist in the DNA to cause toxicity (21, 23, 27, 43). Consistent with these observations, nitrofuran-induced lesions were found to be poor substrates for nucleotide excision repair in vitro (46).Taken together, these observations suggest to us that the cellular response to nitrofurazone will be distinct from its response to UV irradiation. However, no study has examined the relative contributions that nucleotide excision repair, translesion synthesis, or recombination has in recovering from nitrofurazone-induced damage. In this study, we characterized the mechanism by which nitrofurazone inhibits DNA replication and identified the genes that contribute to the recovery, survival, and mutagenesis of Escherichia coli treated with nitrofurazone. In contrast to previous studies, we found that survival following nitrofurazone-induced damage depends predominantly on nucleotide excision repair. Similarly to UV-induced DNA damage, both the RecF and RecBC pathways contribute to survival following nitrofurazone-induced DNA damage. The contribution of translesion polymerases to survival was minor and was mediated by Pol IV. In addition, we found that nitrofurazone can act to inhibit DNA replication directly when used at higher concentrations. The direct inhibition of replication is reversible and occurs independently of DNA damage, suggesting that DNA is not the primary target of its antimicrobial properties.