Selection for Chromosome Architecture in Bacteria

Bacterial chromosomes are immense polymers whose faithful replication and segregation are crucial to cell survival. The ability of proteins such as FtsK to move unidirectionally toward the replication terminus, and direct DNA translocation into the appropriate daughter cell during cell division, requires that bacterial genomes maintain an architecture for the orderly replication and segregation of chromosomes. We suggest that proteins that locate the replication terminus exploit strand-biased sequences that are overrepresented on one DNA strand, and that selection increases with decreased distance to the replication terminus. We report a generalized method for detecting these architecture imparting sequences (AIMS) and have identified AIMS in nearly all bacterial genomes. Their increased abundance on leading strands and decreased abundance on lagging strands toward replication termini are not the result of changes in mutational bias; rather, they reflect a gradient of long-term positive selection for AIMS. The maintenance of the pattern of AIMS across the genomes of related bacteria independent of their positions within individual genes suggests a well-conserved role in genome biology. The stable gradient of AIMS abundance from replication origin to terminus suggests that the replicore acts as a target of selection, where selection for chromosome architecture results in the maintenance of gene order and in the lack of high-frequency DNA inversion within replicores. [Reviewing Editor: Dr. Martin Kreitman]     

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.     

In silico prediction of the origin of replication among bacteria: a case study of Bacteroides thetaiotaomicron

The initiation of chromosomal replication occurs only once during the prokaryote cell cycle. Some origins of replication have been experimentally determined and have led to the development of in silico approaches to find the origin of replication among other prokaryotes. DNA base composition asymmetry is the basis of numerous in silico methods used to detect the origin and terminus of replication in prokaryotes. However, the composition asymmetry does not allow us to locate precisely the positions of the origin and terminus. Since DNA replication is a key step in the cell cycle it is important to determine properly the origin and terminus regions. Therefore, we have reviewed here the methods, tools, and databases for predicting the origins and terminuses of replication, and we have proposed some complementary analyses to reinforce these predictions. These analyses include finding the dnaA gene and its binding sites; making BLAST analyses of the intergenic sequences compared to related species; studying the gene order around the origin sequence; and studying the distribution of the genes encoded in the leading versus the lagging strand.     

Chromosome I Controls Chromosome II Replication in Vibrio cholerae

Control of chromosome replication involves a common set of regulators in eukaryotes, whereas bacteria with divided genomes use chromosome-specific regulators. How bacterial chromosomes might communicate for replication is not known. In Vibrio cholerae, which has two chromosomes (chrI and chrII), replication initiation is controlled by DnaA in chrI and by RctB in chrII. DnaA has binding sites at the chrI origin of replication as well as outside the origin. RctB likewise binds at the chrII origin and, as shown here, to external sites. The binding to the external sites in chrII inhibits chrII replication. A new kind of site was found in chrI that enhances chrII replication. Consistent with its enhancing activity, the chrI site increased RctB binding to those chrII origin sites that stimulate replication and decreased binding to other sites that inhibit replication. The differential effect on binding suggests that the new site remodels RctB. The chaperone-like activity of the site is supported by the finding that it could relieve the dependence of chrII replication on chaperone proteins DnaJ and DnaK. The presence of a site in chrI that specifically controls chrII replication suggests a mechanism for communication between the two chromosomes for replication.     

Comprehensive comparison between locations of orthologous genes on archaeal and bacterial genomes

MOTIVATION: Following an extensive search for orthologous genes between the complete genomes from archaea and bacteria, the spatial association of the orthologs has been investigated in terms of synteny, the conservation of the order of neighboring genes. However, the relationships between the relative locations of remote orthologs over entire genomes have not been shown. RESULTS: Comprehensive comparisons between the locations of orthologs on nineteen archaeal and bacterial genomes are presented by the location to location correspondence based on the gene-location distance. When the two genomes are rotated such that a pair of orthologs with the shortest distance is set in the same angle, a statistically significant number of orthologs maintain their relative locations between the genomes. Even by the short distances at the 5% significance level, the rotations are restricted within a narrow range, suggesting an intrinsic angle for realizing similar locations between the orthologs in each genome pair. Furthermore, the rotations in the restricted range agree with the replication origin and terminus sites for the analyzed genomes where such sites are known. The relationship between location-maintained orthologs and gene function is also discussed.     

Noise-reduction filtering for accurate detection of replication termini in bacterial genomes

Bacterial chromosomes are highly polarized in their nucleotide composition through mutational selection related to replication. Using compositional skews such as the GC skew, replication origin and terminus can be predicted in silico by observing the shift points. However, the genome sequence is affected by myriad functional requirements and selection on numerous subgenomic features, and elimination of this "noise" should lead to better predictions. Here, we present a noise-reduction approach that uses low-pass filtering through Fast Fourier transform coupled with cumulative skew graphs. It increases the prediction accuracy of the replication termini compared with previously documented methods based on genomic base composition.     

Regulation of Chromosome Replication in Bacillus subtilis: Effects of Amino Acid Starvation in Strain 168

Regulation of chromosome replication in Bacillus subtilis strain 168, in response to starvation for an essential amino acid, was found to differ from that reported for Escherichia coli. Not all replication points stop at the terminus during amino acid starvation. There is some evidence, however, to indicate that preferred stopping sites might exist. Initiation at the origin can occur in the absence of total protein synthesis as well as when the deoxyribonucleic acid (DNA)- mass ratio is unbalanced. DNA synthesis appears to be controlled independently of the initiation event by a second regulatory circuit, that may utilize the DNA-mass ratio. Once initiated, chromosome replication does not always go to completion in an uninterrupted sequence.     

Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not

Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.     

Viral genomes maintained extrachromosomally in hamster polyomavirus-induced lymphomas display a cell-specific replication in vitro.

Hamster polyomavirus causes lymphomas when injected into newborn Syrian hamsters. Large amounts of extrachromosomal viral genomes are accumulated in the lymphoma cells. These genomes are characterized by deletions affecting the late coding region as well as a specific part of the noncoding regulatory region. By contrast with wild-type genomes, lymphoma-associated genomes replicate in a lymphoblastoid cell line but not in a fibroblastic cell line. The deletion acts in a cis-dominant manner and is the primary determinant of this host-range effect on replication. The boundaries of the regulatory region necessary for viral DNA replication in the two cell contexts have been defined. The regulatory region can be functionally divided in two domains: one domain (distal from the origin of replication) is necessary for viral genome replication in fibroblasts, whereas the other domain (proximal to the origin of replication) is functional only in the lymphoblastoid cell context and contains the sequence specifically conserved in the lymphoma-associated genomes. This sequence harbors a motif recognized by a lymphoblastoid cell-specific trans-acting factor.     

A dimeric Rep protein initiates replication of a linear archaeal virus genome: implications for the Rep mechanism and viral replication

The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between an active-site tyrosine and the 5' end of the DNA, releasing a 3' DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral replication are discussed.     

Physical and genetic mapping of mammalian replication origins.

The neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques are sensitive physical mapping methods that have been used successfully to identify replication initiation sites in genomes of widely varying complexity. We present detailed methodology for the preparation of replication intermediates from mammalian cells and their analysis by both neutral/neutral and neutral/alkaline two-dimensional gel approaches. The methods described allow characterization of the replication pattern of single-copy loci, even in mammalian cells. When applied to metazoans, initiation is found to occur at multiple sites scattered throughout zones that can be as long as 50 kb, with some subregions being preferred. Although these observations do not rule out the possibility of genetically defined replicators, they offer the alternative or additional possibility that chromosomal context may play an important role in defining replication initiation sites in complex genomes. We discuss novel recombination strategies that can be used to test for the presence of sequence elements critical for origin function if the origin lies in the vicinity of a selectable gene. Application of this strategy to the DHFR locus shows that loss of sequences more than 25 kb from the local initiation zone can markedly affect origin activity in the zone.     

Mode of replication of the conjugative R-plasmid RSF1040 in Escherichia coli.

Replicating deoxyribonucleic acid (DNA) molecules of plasmid RSF1040, a deletion mutant of the conjugative R plasmid R6K, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency. The partially supercoiled molecules sediment more rapidly than native covalently closed circular DNA in neutral sucrose gradients and band at a position intermediate between covalently closed circular and open circular DNA in CsClethidium bromide gradients. Electron microscope measurements of the linear EcoRI-treated replicative intermediates indicate that replication can be initiated at two sites (origins) on the plasmid DNA molecule located at about 23% (alpha) and 39% (beta) of the total genome length from an EcoRI end designated arbitrarily as the "left-hand" end of the molecule. The overall replication of RSF1040 is asymmetrically bidirectional. Replication from the alpha origin proceeds first to the "right" to a unique termination site located some 55% of the total genome length from the left-hand end of the molecule. At this point replication proceeds from the alpha origin to the "left" (i.e., opposite to the original direction of replication) until replication of the molecule is completed. Replication also proceeds from the beta origin asymmetrically to the unique terminus site.     

The replication fork trap and termination of chromosome replication

Bacteria that have a circular chromosome with a bidirectional DNA replication origin are thought to utilize a ‘replication fork trap’ to control termination of replication. The fork trap is an arrangement of replication pause sites that ensures that the two replication forks fuse within the terminus region of the chromosome, approximately opposite the origin on the circular map. However, the biological significance of the replication fork trap has been mysterious, as its inactivation has no obvious consequence. Here we review the research that led to the replication fork trap theory, and we aim to integrate several recent findings that contribute towards an understanding of the physiological roles of the replication fork trap. Likely roles include the prevention of over‐replication, and the optimization of post‐replicative mechanisms of chromosome segregation, such as that involving FtsK in Escherichia coli.     

Attachment of the chromosome to the cell poles: the strategy for the growth of bacteria in two and three dimensions.

Bacteria such as Staphylococcus, Lampropedia, and Sarcina develop in characteristic two-or three-dimensional groups of cells. We propose here a model of how bacteria may generate such groupings by an extension of an earlier model for rod-shaped bacteria. No other mechanism for forming two- or three-dimensional structures of groups of cells has been proposed. Our earlier model for division of rod-shaped bacteria into nearly equal-sized daughters assumed that the origin and terminus DNA were attached at a critical time to polar wall sites. While such binding was speculative 20 years ago, it has now been established that the DNA for the origin of replication, at least during some part of the cell cycle is located in the pole for several different bacteria. Evidence is also building showing that the terminus DNA region is sometimes located at a position in the cell that will develop into two new poles. Here, a new extension of the concept that polar sites bind specifically origin and terminus DNA of the chromosome is presented that can explain how division takes place in one and then in another dimension to form two-dimensional tablets of four cells or large planar arrays. A further possible extension to three dimensions to generate octets of cells is proposed.     

Single molecule analysis of replicated DNA reveals the usage of multiple KSHV genome regions for latent replication

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences.