Replication of kinetoplast DNA: an update for the new millennium

In this review we will describe the replication of kinetoplast DNA, a subject that our lab has studied for many years. Our knowledge of kinetoplast DNA replication has depended mostly upon the investigation of the biochemical properties and intramitochondrial localisation of replication proteins and enzymes as well as a study of the structure and dynamics of kinetoplast DNA replication intermediates. We will first review the properties of the characterised kinetoplast DNA replication proteins and then describe our current model for kinetoplast DNA replication.     

Specific initiation at an origin of replication from Schizosaccharomyces pombe.

Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals.     

DNA polymerases β and λ and their roles in DNA replication and repair

DNA-dependent DNA polymerases are the main enzymes that catalyze DNA replication. Higher eukaryotic cells have 19 DNA polymerases with strikingly different properties [1]. Mitochondrial DNA polymerase γ of the A family and most of the nuclear enzymes of the B family are high-fidelity DNA polymerases that are involved not only in genomic DNA replication but also in DNA repair. Among the other 15 proteins, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are also involved in repair, including base excision repair and nonhomologous end joining. Some of them play a specific role in replication of damaged DNA templates. This process is referred to as translesion synthesis (TLS). DNA polymerases β and λ, which belong to the X structural family, are polyfunctional enzymes; their properties and functions are discussed.     

Analysis of the kinetic hairpin transfer model for parvoviral DNA replication

All linear DNA molecules face special problems in replicating their 5' ends, as DNA polymerases add nucleotides only to pre-existing strands with free 3'-OH groups. Parvoviruses, a group of small animal viruses with a linear single-stranded DNA genome, cope with this problem by having palindromic terminal sequences that can fold back on themselves to form hairpin structures essential in priming DNA replication. The 3' terminal sequence that initiates replication becomes reversed in orientation during the process, and if the palindrome is imperfect, two different, reverse-complementary terminal sequences are generated. The relative abundances of the terminal sequence orientations at each end of the DNA molecules can be measured and give information about the replication process. From such clues, we developed a "kinetic hairpin transfer model" based on differential rates of hairpin formation and inversion processes depending on the conformations of the 3' termini. Numerical studies showed that this simple idea can account for the diverse pattern of DNA distributions observed in the family Parvoviridae. In this paper, we simplify the model to a set of coupled linear first-order ordinary differential equations in order to delineate its essential properties by Perron-Frobenius theory. Secondly, we examine our assumption of linear kinetics by modeling enzyme catalysis of the component steps of the hairpin transfer process. We show that the rate-determining step of the process is the binding of initiation complex to the self-priming hairpin structures. Furthermore, we find that if the replication machinery is saturated by DNA substrate late in an infection, the differential equations become non-linear but the steady-state DNA distribution is still given by the solution of our original linear equations.     

A rotary pumping model for helicase function of MCM proteins at a distance from replication forks

We propose an integrated model for eukaryotic DNA replication to explain the following problems: (1) How is DNA spooled through fixed sites of replication? (2) What and where are the helicases that unwind replicating DNA? (3) Why are the best candidates for replicative helicases, namely mini-chromosome maintenance (MCM) proteins, not concentrated at the replication fork? (4) How do MCM proteins spread away from loading sites at origins of replication? We draw on recent discoveries to argue that the MCM hexameric ring is a rotary motor that pumps DNA along its helical axis by simple rotation, such that the movement resembles that of a threaded bolt through a nut, and we propose that MCM proteins act at a distance from the replication fork to unwind DNA. This model would place DNA replication in a growing list of processes, such as recombination and virus packaging, that are mediated by ring-shaped ATPases pumping DNA by helical rotation.     

DNA replication in eukaryotes: a model for the specific involvement of chromatin subunits.

A model is proposed whereby eukaryotic DNA replication is specifically directed by the 200 base pair repeat structure of the DNA-histone complex. The model proposes a mechanism for the sequential, bidirectional replication of DNA from initial origin points on the chromatin fibre and is consistent with the known properties of eukaryotic DNA replication. Several predictions can be made from the model which are amenable to testing.     

Binding properties of replication protein A from human and yeast cells.

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.     

Recombinational repair and restart of damaged replication forks

Genome duplication necessarily involves the replication of imperfect DNA templates and, if left to their own devices, replication complexes regularly run into problems. The details of how cells overcome these replicative 'hiccups' are beginning to emerge, revealing a complex interplay between DNA replication, recombination and repair that ensures faithful passage of the genetic material from one generation to the next.     

Initiation of DNA Replication in Cells of Higher Eukaryotes

In this review, the problems concerning initiation of DNA replication in higher eukaryotes are discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.     

Initiation of DNA replication in higher eukaryotes

In this review, of problems concerning initiation of DNA replication in higher eukaryotes is discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.     

Replication origins in yeast chromosomes

DNA replication initiates at many sites in eukaryotic chromosomes. It has been difficult to isolate such replication origins, but a family of sequences from the yeast genome have properties which suggest that they may serve this function. The identification of these sequences together with sophisticated methods of genetic analysis, make yeast a useful organism for the study of eukaryotic DNA replication.     

Replication timing properties across the pseudoautosomal region boundary and cytogenetic band boundaries on human distal Xp

The establishment of human chromosomal regions as distinct and characteristic domains has been demonstrated by the reproducible banding patterns observed on metaphase chromosomes as a result of various staining techniques. Although the exact molecular properties responsible for the patterns are not well understood, a general correlation has been established between the time of replication of a particular region of DNA and its banding characteristics. Using a replication timing assay based on fluorescence in situ hybridization patterns, we investigated replication timing properties across chromosomal regions with potentially distinct chromatin properties. Relative replication timing values were determined using cosmid DNA probes around the pseudoautosomal region boundary in Xp22.3 and the cytogenetic band boundary regions surrounding Xp22.2. Although we observed replication timing domains that were generally consistent with cytogenetic banding patterns, we did not find sharp replication timing boundaries at either the pseudoautosomal region boundary or at the cytogenetic band boundaries. Received: 6 September 1997; in revised form: 16 December 1997 / Accepted: 5 January 1998     

Herpes simplex virus type 1 ICP8: helix-destabilizing properties.

The major single-stranded DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is one of seven virus-encoded polypeptides required for HSV-1 DNA replication. To investigate the role of ICP8 in viral DNA replication, we have examined the interaction of ICP8 with partial DNA duplexes and found that it can displace oligonucleotides annealed to single-stranded M13 DNA. In addition, ICP8 can melt small fragments of fully duplex DNA. Unlike a DNA helicase, ICP8-promoted strand displacement is ATP and Mg2+ independent and exhibits no directionality. It requires saturating amounts of ICP8 and is both efficient and highly cooperative. These properties make ICP8 suitable for a role in DNA replication in which ICP8 destabilizes duplex DNA during origin unwinding and replication fork movement.     

Directly repeated, 20-bp sequence of plasmid R1162 DNA is required for replication, expression of incompatibility, and copy-number control

DNA required in cis for the replication of the broad-host-range plasmid R1162 is located on two contiguous HpaII fragments of 210 and 370 bp. The latter of these contains three and one-half, perfectly conserved, 20-bp directly repeated sequences. The significance of these for plasmid replication, incompatibility, and copy-number control was examined by generating deletions into these repeats and testing the properties of the remaining DNA. We conclude from the results that the direct repeats are essential for expression of incompatibility and for the decrease in copy number observed when the directly repeated DNA is cloned into R1162. Little, if any, additional DNA is required from the ori region for these properties. Moreover, deletions of intermediate size result in an intermediate level of incompatibility, indicating the importance of the periodic structure of the direct repeats. The directly repeated DNA is also required for an active origin of replication, as are additional, nonrepeated sequences adjacent to this DNA. The properties of the direct repeats are discussed with respect to their possible role in the replication of R1162 DNA.     

DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum

Two-dimensional neutral/neutral agarose gel electrophoresis is used extensively to localize replication origins. This method resolves DNA structures containing replication forks. It also detects X-shaped recombination intermediates in meiotic cells, in the form of a typical vertical spike. Intriguingly, such a spike of joint DNA molecules is often detectable in replicating DNA from mitotic cells. Here, we used naturally synchronous DNA samples from Physarum polycephalum to demonstrate that postreplicative, DNA replication-dependent X-shaped DNA molecules are formed between sister chromatids. These molecules have physical properties reminiscent of Holliday junctions. Our results demonstrate frequent interactions between sister chromatids during a normal cell cycle and suggest a novel phase during DNA replication consisting of transient, joint DNA molecules formed on newly replicated DNA.     

Processing of plasmid DNA with ColE1-like replication origin

With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.