Nonrandom clusters of palindromes in herpesvirus genomes.

Palindromes are symmetrical words of DNA in the sense that they read exactly the same as their reverse complementary sequences. Representing the occurrences of palindromes in a DNA molecule as points on the unit interval, the scan statistics can be used to identify regions of unusually high concentration of palindromes. These regions have been associated with the replication origins on a few herpesviruses in previous studies. However, the use of scan statistics requires the assumption that the points representing the palindromes are independently and uniformly distributed on the unit interval. In this paper, we provide a mathematical basis for this assumption by showing that in randomly generated DNA sequences, the occurrences of palindromes can be approximated by a Poisson process. An easily computable upper bound on the Wasserstein distance between the palindrome process and the Poisson process is obtained. This bound is then used as a guide to choose an optimal palindrome length in the analysis of a collection of 16 herpesvirus genomes. Regions harboring significant palindrome clusters are identified and compared to known locations of replication origins. This analysis brings out a few interesting extensions of the scan statistics that can help formulate an algorithm for more accurate prediction of replication origins.     

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.     

Identification of replication origins in archaeal genomes based on the Z-curve method

The Z-curve is a three-dimensional curve that constitutes a unique representation of a DNA sequence, i.e., both the Z-curve and the given DNA sequence can be uniquely reconstructed from the other. We employed Z-curve analysis to identify one replication origin in the Methanocaldococcus jannaschii genome, two replication origins in the Halobacterium species NRC-1 genome and one replication origin in the Methanosarcina mazei genome. One of the predicted replication origins of Halobacterium species NRC-1 is the same as a replication origin later identified by in vivo experiments. The Z-curve analysis of the Sulfolobus solfataricus P2 genome suggested the existence of three replication origins, which is also consistent with later experimental results. This review aims to summarize applications of the Z-curve in identifying replication origins of archaeal genomes, and to provide clues about the locations of as yet unidentified replication origins of the Aeropyrum pernix K1, Methanococcus maripaludis S2, Picrophilus torridus DSM 9790 and Pyrobaculum aerophilum str. IM2 genomes.     

Identification of replication origins in the genome of the methanogenic archaeon,<Emphasis Type="Italic"> Methanocaldococcus jannaschii</Emphasis>

Methanocaldococcus jannaschii has been notorious as an archaeon in which the replication origins are difficult to identify. Although extensive efforts have been exerted on this issue, the locations of replication origins still remain elusive 7 years after the publication of its complete genome sequence in 1996. Ambiguous results were obtained in identifying the replication origins of M. jannaschii based on all theoretical and experimental approaches. In the genome of M. jannaschii, we found that an ORF (MJ0774), annotated as a hypothetical protein, is a homologue of the Cdc6 protein. The position of the gene is at a global minimum of the x component of the Z curve, i.e., RY disparity curve, which has been used to identify replication origins in other Archaea. In addition, an intergenic region (694,540–695,226 bp) that is between the cdc6 gene and an adjacent ORF shows almost all the characteristics of known replication origins, i.e., it is highly rich in AT composition (80%) and contains multiple copies of repeat elements and AT stretches. Therefore, these lines of evidence strongly suggest that the identified region is a replication origin, which is designated as oriC1. The analysis of the y component of the Z curve, i.e., MK disparity curve, suggests the presence of another replication origin corresponding to one of the peaks in the MK disparity curve at around 1,388 kb of the genome.Communicated by G. Antranikian     

Machinery to support genome segment inversion exists in a herpesvirus which does not naturally contain invertible elements

In many herpesviruses, genome segments flanked by inverted repeats invert during DNA replication. It is not known whether this inversion is a consequence of an inherently recombinagenic replicative mechanism common to all herpesviruses or whether the replication enzymes of viruses with invertible segments have specifically evolved additional enzymatic activities to drive inversion. By artificially inserting a fusion of terminal sequences into the genome of a virus which normally lacks invertible elements (murine cytomegalovirus), we created a genome composed of long and short segments flanked by 1,359- and 543-bp inverted repeats. Analysis of genomic DNA from this virus revealed that inversion of both segments generates equimolar amounts of four isomers during the viral propagation necessary to produce DNA for analysis from a single viral particle. We conclude that a herpesvirus which naturally lacks invertible elements is able to support efficient segment inversion. Thus, the potential to invert is probably inherent in the replication machinery of all herpesviruses, irrespective of genome structure, and therefore genomes with invertible elements could have evolved simply by acquisition of inverted repeats and without concomitant evolution of enzymatic activities to mediate inversion. Furthermore, the recombinagenicity of herpesvirus DNA replication must have some importance independent of genome segment inversion.     

Modeling inhomogeneous DNA replication kinetics

In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.     

Physical mapping of origins of replication in the fission yeast Schizosaccharomyces pombe.

We isolated four fragments from the Schizosaccharomyces pombe genome that mediate autonomous replication. A two-dimensional gel analysis revealed that in each case initiation could be mapped to within the S. pombe sequences. In three of the fragments, initiation could be mapped to one discrete location. In the fourth fragment, subcloning and two-dimensional gel analysis suggested that two discrete origins of replication were located within 3 kb of each other. When in proximity, usually only one of these origins fired, suggesting origin interference. Two-dimensional gel analysis of the four origin fragments at their genomic locations demonstrated that each is used in the chromosomes, but in only a subset of cells or cell divisions. The S. pombe genome appears to contain many discrete origins, not all of which fire in any given cell and some of which are closely spaced. Not I/Sfi I mapping of the five origins from this and a previous study indicates that they are randomly distributed throughout the genome and appear to be representative of chromosomal origins of replication in this organism. We compare the features of S. pombe replication origins with those of S. cerevisiae and animal cells.     

Compositional Bias is a Major Determinant of the Distribution Pattern and Abundance of Palindromes in Drosophila melanogaster

Palindromic sequences are important DNA motifs related to gene regulation, DNA replication and recombination, and thus, investigating the evolutionary forces shaping the distribution pattern and abundance of palindromes in the genome is substantially important. In this article, we analyzed the abundance of palindromes in the genome, and then explored the possible effects of several genomic factors on the palindrome distribution and abundance in Drosophila melanogaster. Our results show that the palindrome abundance in D. melanogaster deviates from random expectation and the uneven distribution of palindromes across the genome is associated with local GC content, recombination rate, and coding exon density. Our data suggest that base composition is the major determinant of the distribution pattern and abundance of palindromes and the correlation between palindrome density and recombination is a side-product of the effect of compositional bias on the palindrome abundance.     

The dynamics of genome replication using deep sequencing

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology.     

Methylation is co-ordinated on the putative replication origins of Physarum ribosomal DNA

In Physarum polycephalum, the ribosomal DNA is found as 60,000 base-pair palindromes. Each rDNA has four symmetrically arranged replication origins flanked by ribosomal RNA genes. A particular sequence, the putative replication origin, is repeated at the approximate position of each origin and nowhere else in the molecule. On a typical rDNA molecule, only one origin is active per replication cycle. We show that both the level and co-ordination of methylation result in asymmetrically methylated rDNA molecules that are particularly hypomethylated at one of their four putative replication origins. This pattern of methylation on a typical rDNA molecule is consistent with a model where hypomethylation is a determinant of origin activity.     

Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast

DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2'-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains.     

AT excursion: a new approach to predict replication origins in viral genomes by locating AT-rich regions

Background  

Replication origins are considered important sites for understanding the molecular mechanisms involved in DNA replication. Many computational methods have been developed for predicting their locations in archaeal, bacterial and eukaryotic genomes. However, a prediction method designed for a particular kind of genomes might not work well for another. In this paper, we propose the AT excursion method, which is a score-based approach, to quantify local AT abundance in genomic sequences and use the identified high scoring segments for predicting replication origins. This method has the advantages of requiring no preset window size and having rigorous criteria to evaluate statistical significance of high scoring segments.     

Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model

Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.     

Genome-wide characterization of fission yeast DNA replication origins

Eukaryotic DNA replication is initiated from multiple origins of replication, but little is known about the global regulation of origins throughout the genome or in different types of cell cycles. Here, we identify 401 strong origins and 503 putative weaker origins spaced in total every 14 kb throughout the genome of the fission yeast Schizosaccharomyces pombe. The same origins are used during premeiotic and mitotic S-phases. We found that few origins fire late in mitotic S-phase and that activating the Rad3 dependent S-phase checkpoint by inhibiting DNA replication had little effect on which origins were fired. A genome-wide analysis of eukaryotic origin efficiencies showed that efficiency was variable, with large chromosomal domains enriched for efficient or inefficient origins. Average efficiency is twice as high during mitosis compared with meiosis, which can account for their different S-phase lengths. We conclude that there is a continuum of origin efficiency and that there is differential origin activity in the mitotic and meiotic cell cycles.     

Nonparametric methods for analyzing replication origins in genomewide data

Due to the advent of high-throughput genomic technology, it has become possible to monitor cellular activities on a genomewide basis. With these new methods, scientists can begin to address important biological questions. One such question involves the identification of replication origins, which are regions in the chromosomes where DNA replication is initiated. One hypothesis is that their locations are nonrandom throughout the genome. In this article, we analyze data from a recent yeast study in which candidate replication origins were profiled using cDNA microarrays to test this hypothesis. We find no evidence for such clustering.     

Replication fork stalling by bulky DNA damage: localization at active origins and checkpoint modulation

The integrity of the genome is threatened by DNA damage that blocks the progression of replication forks. Little is known about the genomic locations of replication fork stalling, and its determinants and consequences in vivo. Here we show that bulky DNA damaging agents induce localized fork stalling at yeast replication origins, and that localized stalling is dependent on proximal origin activity and is modulated by the intra-S-phase checkpoint. Fork stalling preceded the formation of sister chromatid junctions required for bypassing DNA damage. Despite DNA adduct formation, localized fork stalling was abrogated at an origin inactivated by a point mutation and prominent stalling was not detected at naturally-inactive origins in the replicon. The intra-S-phase checkpoint contributed to the high-level of fork stalling at early origins, while checkpoint inactivation led to initiation, localized stalling and chromatid joining at a late origin. Our results indicate that replication forks initially encountering a bulky DNA adduct exhibit a dual nature of stalling: a checkpoint-independent arrest that triggers sister chromatid junction formation, as well as a checkpoint-enhanced arrest at early origins that accompanies the repression of late origin firing. We propose that the initial checkpoint-enhanced arrest reflects events that facilitate fork resolution at subsequent lesions.     

Nonparametric inference for local extrema with application to oligonucleotide microarray data in yeast genome

Identifying local extrema of expression profiles is one primary objective in some cDNA microarray experiments. To study the replication dynamics of the yeast genome, for example, local peaks of hybridization intensity profiles correspond to putative replication origins. We propose a nonparametric kernel smoothing (NKS) technique to detect local hybridization intensity extrema across chromosomes. The novelty of our approach is that we base our inference procedures on equilibrium points, namely those locations at which the first derivative of the intensity curve is zero. The proposed smoothing technique provides both point and interval estimation for the location of local extrema. Also, this technique can be used to test for the hypothesis of either one or multiple suspected locations being the true equilibrium points. We illustrate the proposed method on a microarray data set from an experiment designed to study the replication origins in the yeast genome, in that the locations of autonomous replication sequence (ARS) elements are identified through the equilibrium points of the smoothed intensity profile curve. Our method found a few ARS elements that were not detected by the current smoothing methods such as the Fourier convolution smoothing.