Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB 6 –DnaC 6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB 6 –DnaC 6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB 6 –DnaC 6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine
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Replication initiation sites are discrete chromosomal loci defined by the binding of the ORC and the Mcm2-7 complex. By contrast, replication termination events are widely distrib- uted (Figure 4). In a population of cells, the majority of the genome (>75%; Figure 4C) is within 1 kb of a termination event in at least 1% of cells. For context, the most probable termina- tion sites will only experience a termination event per kb in 3.9% of cells. The distribution of termination sites that we find is consistent with and greatly extends previously reported termi- nation (TER) sites (Fachinetti et al., 2010). Those TER sites were found to colocate with particular chromosomal features, including tRNAs and centromeres. However, our observation that these sites constitute only a small minority of all termination events, combined with observations that such sites do not pause replication forks (Azvolinsky et al., 2009), suggest that they do not directly influence termination, although we note that the current available resolution does not rule out the possi- bility that particular sequence properties might influence the precise location of termination events. Perturbing genome repli- cation, by the inactivation of three active replication origins, tested how termination sites are specified. The inactivated origins did not globally alter the pattern of genome replication, allowing investigation of the consequences for termination. The inactivations resulted in changes in the location of termina- tion sites, consistent with origin activity being the principal influence over the distribution. Recently reported genome- wide perturbations to DNA replication origin activity also resulted in relocalization of termination events (McGuffee et al., 2013). These findings are consistent with the observed distribution of base substitutions in a mutator strain, which were hypothesized to be a consequence of considerable vari- ability in fork termination sites (Larrea et al., 2010). We conclude that the diversity in termination sites can be explained by the
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It is common for initiation to have occurred in the previous generation even under conditions of slow growth (i.e., the total of the combined C and D periods [C ⫹ D] is greater than the doubling time [ ]). Yet cells rarely exhibit multifork replication. In multifork replication, initiation begins before the previous termina- tion event completes, such that a single replicating chromosome possesses four or even eight copies of ori. Instead, B. subtilis normally initiates when the cell contains complete, homologous chromosomes where the copy number represents a power of 2. In fact, replication initiation often proceeds immediately after the previous termination event. This may be due to the role of YabA in B. subtilis replication initiation control, which ties DnaA activity to DnaN availability (27, 28). Multifork replication is comparatively common in E. coli, where Hda is thought to play a similar but mechanistically distinct role in reducing initiation potential during ongoing replication (7, 29).
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While knock-out of PR-Set7 is lethal in mammalians, absence of both Suv4-20h1 and -h2 displays only minor cell cycle defects (Oda et al. 2010; S. Wu and Rice 2011; Beck et al. 2012; Schotta et al. 2008). Indeed, genome-wide timing experiments only revealed a delay of some late replicating domains when Suv4-20h1/2 were missing. This indicates that while replication origin licensing and activation is perturbed in absence of H4K20me2/3, the concerned regions are still replicated, most likely passively. These results imply that the severe phenotype of PR-Set7 knock-out presumably depend on other functions than replication regulation. It has recently been reported, that unmethylated H4K20 (H4K20me0) marks post-replicative chromatin in G2 and is read by the H3-H4 histone chaperone TONSL-MMS22L, which is implicated in DNA repair (Saredi et al. 2016). Taken together, this would situate H4K20me0/1 and PR-Set7 as cell cycle sensor mechanism, with H4K20me0 marking post-replicative chromatin and H4K20me1 identifying G1 chromatin, primed for replication. This scenario would explain both the re-replication phenotype when PR-Set7 is stabilized – aberrant H4K20me1 in G2 mimics G1 cell cycle stage prone for replication – and cell cycle arrest in absence of PR-Set7 – as the cell cannot distinguish between pre- and post-replicative chromatin. Consequently, H4K20me1 might serve as cell cycle stage marker, but is not necessarily directly linked to origin licensing and replication initiation.
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using PI-stained cells). The RNA samples were then subjected to microarray analysis as described in Materials and Methods. We found a total of 855 genes induced in E1A-expressing cells com- pared to that of Adb-gal-infected cells (fold change cutoff ⬎ 1.5; P ⬍ 0.05). Gene ontology analysis indicated that a large number of these genes belonged to various pathways that lead to cell cycle progression, including initiation and elongation of DNA replica- tion, and that several genes are involved in the DNA damage re- sponse (a partial list of these genes is shown in Table S1 and Table S2 in the supplemental material). These results are in broad agree- ment with two other published reports that studied the E1A-in- duced genes in serum-starved cells (37, 38). To confirm the induc- tion of these genes and to get an idea as to how the induction by E1A compares with that of serum-stimulated cells, induction of 12 of the genes was compared to induction of those induced in se- rum-stimulated MCF10A cells using qPCR assays. As shown in Fig. 1A, all of the replication initiation genes tested in these exper- iments (Cdt1, Cdc6, MCM3, MCM4, GINS2, RFC5, and Cdk2; see reference 15) and genes involved in other aspects of DNA replication (DNMT1, PolE2, PCNA) and cell cycle progression genes (CCNE2 or Cyclin E2 and E2F2) were induced at levels significantly higher than those seen with serum-stimulated cells. The magnitude of induction was quite variable, ranging from 2-fold (PCNA) to 6-fold (Cdt1). It is interesting that of the E2F family members, only E2F2 was induced in these experiments. The significance of E2F2 induction in E1A-induced cell cycle pro- gression is not clear.
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Matched deletions or additions at complementary termini suggest that the RNAs either are copied from each other during replication (end-to-end replication) or are continuously synthesized from full-length templates. The latter possibility would require the use of multiple replication initiation and termination sites in the genomic and antigenomic RNAs. Ini- tiation sites would have to be at the same position as the termination sites on the opposite, template, strand in order for subsequent rounds of replication to generate deletions of the same size on the complementary genomic and antigenomic termini. Furthermore, during persistence, use of internal sites would have to increase over the use of terminal sites to result in a rise in the concentration of terminally truncated RNAs. Although these events could occur, the alternate possibility, that truncated RNAs may be capable of replication, has more support: terminally truncated RNAs are found only in nucleo- capsids containing replicating RNAs and are not found in the nonreplicating mRNA fraction of the cell; terminally truncated RNAs are packaged into virions, and when virions containing RNAs with terminal deletions are used to infect mice or BHK-21 cells, the same-size deletions are present after infec- tion and the deletions are maintained (33, 34); and genomes of other viruses having terminal nucleotide additions or deletions are known to be faithfully replicated (27, 47). Therefore, the most likely interpretation is that terminally truncated RNAs are copied from each other during replication and that initia- tion occurs at the termini of all or most of the RNAs present in the nucleocapsid: those with deleted termini, those with extended termini, and those with normal termini.
Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.
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The molecular mechanism of T4 replication initiation has been investigated in vitro using R-loop substrates constructed by annealing an RNA oligonucleotide to supercoiled oriF plasmids . Efficient replication of these preformed R-loop substrates does not require a promoter sequence, but a DUE is necessary. In fact, non-origin plasmids are efficiently replicated in vitro by the T4 replisome as long as they have a preformed R- loop within a DUE region, implying that the R-loop itself is the signal for replisome assembly on these sub- strates. Experiments using radioactively labeled R-loop RNA directly demonstrated that the RNA is used as the primer for DNA synthesis. Several viral proteins are required for significant replication of these R-loop sub- strates: DNA polymerase (gp43), polymerase clamp (gp45), clamp loader (gp44/62), and single-stranded DNA binding protein (gp32). In addition, without the replicative helicase (gp41), leading-strand synthesis is limited to a relatively short region (about 2.5 kb) and lagging strand synthesis is abolished. While gp41 can load without the helicase loading protein (gp59), the presence of gp59 greatly accelerates the process. Finally, replication on these covalently closed substrates is severely limited when the T4-encoded type II topoi- somerase (gp39/52/60) is withheld, as expected due to the accumulation of positive supercoiling ahead of the fork.
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The proper inheritance of genomic information in eukaryotes requires both well-coordinated DNA replica- tion in S phase and separation of duplicated chromo- somes into daughter cells in mitosis . Prior to S phase, pre-replication complex (pre-RC), a multi-protein complex which dictates when and where the DNA repli- cation will initiate, is assembled [2-6]. Studies in Sac- charomyces cerevisiae revealed conserved replication initiation sites (origins) that comprise a highly conserved autonomously replicating sequence (ARS) . Identifica- tion of proteins bound to this sequence led to the dis- covery of a six-subunit complex that serves as the initiator to select replication initiation sites, and was therefore named the origin recognition complex (ORC) . The assembly of pre-RC starts with ORC recogniz- ing the replication elements and recruiting two factors, Cdc6 and Cdt1. These proteins function together to load the minichromosome maintenance proteins (MCM) onto chromatin [2-6]. This process takes place as early as the end of mitosis of the previous cell cycle . In yeast, at the onset of S phase, Dbf4-dependent kinase
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Obtaining functionally intact modified replication initiator proteins of ssDNA viruses is difficult since, in the case of FBNYV, alteration of two amino acids at the N or C terminus of the M-Rep protein already abolishes its activity. The impor- tance of methionine 1 and arginine 3 for M-Rep is reflected by the fact that 6H-MRep-S is not functional, whereas 6H- MRep-M in which, apart from the tag, the wild-type amino acid sequence is conserved catalyzes replication initiation in planta. The lower replication level of the replicon R-6H-M compared to wild-type DNA-R (Fig. 2) indicates that the oli- gohistidine tag impairs to some extent the function of the protein. The replacement of methionine 1 by alanine in 6H- MRep-A or 4H-MRep-A results in a further reduction of DNA replication. Whether the reduced activity of 6H- MRep-A and 4H-MRep-A is solely due to the methionine 1 to alanine change or whether it is also influenced by the oligohis- tidine tag immediately preceding methionine 1 remains to be determined. Basic amino acids at the N terminus of the TYLCSV Rep have been suggested to be implicated in DNA recognition by the protein (8), an idea in line with the results presented here. An alternative or additional explanation for the observed reduction of DNA replication efficiency may be that important DNA-R elements required in cis were located in the sequence immediately preceding and/or encoding the amino terminus of M-Rep. The addition of the 27 nucleotides of the tag and base changes within the following sequence may interfere with the correct function of such cis-acting elements. Experiments uncoupling M-Rep expression from cognate DNA (template) replication will provide distinctive information.
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Despite this, a number of parallels can be drawn between the regulatory mechanisms in the two species. Both organisms utilize a major regulator during vegetative growth which interacts with both DnaA and DnaN; YabA in B. subtilis  and Hda in E. coli . This may provide the respective species with a mechanism for appropriately timing initiation of replication, since DnaN is a key component of the DNA elongation complex. Both organisms have regulators which are implicated in chromosome segregation, SeqA in E. coli, and Soj and SirA during growth and sporulation of B. subtilis, respectively. Both organisms appear to utilize a method of origin sequestration to prevent DnaA binding: in E. coli, SeqA binds to newly replicated origins, and in B. subtilis Spo0A~P is able to bind to the origin, playing an albeit more modest role in inhibiting DNA replication. Furthermore, both organisms have evolved a regulator which targets a structurally equivalent location on DnaA domain I—the sporulation inhibitor of replication in B. subtilis, SirA, and the promoter of DNA replication initiation in E. coli, DiaA. Thus, these may represent common themes of replication regulation across bacterial species.
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Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. One of the major roles of CDK is to promote chromosomal DNA replication. However, how CDKs promote DNA replication has been a long-standing question, because all the essential CDK substrates in DNA replication have not been identified yet. Recently Sld2 and Sld3 were identified as essential substrates of CDKs in the initiation step of DNA replication in budding yeast. Moreover, bypass of their phosphorylations is sufficient to promote DNA replication. Phosphorylation of Sld2 and Sld3 by CDKs enhances the formation of complex(es) with a BRCT (BRCA1 C-Terminal)-containing replication protein, Dpb11. We further propose that multiple phosphorylation by CDKs controls this process in budding yeast. Even though Sld3 orthologues in multicellular eukaryotes have not been identified, similar complex formation and, therefore, a similar mechanism of initiation control might be employed in eukaryotes.
complete replication of RNA could result because the prepa- ration lacks a cofactor necessary for plus-strand synthesis. It has recently been demonstrated that the La protein in mos- quito cells and chicken cells binds to the 39 end of SIN minus- strand RNA with high affinity (24, 25), and it has been postu- lated that this protein may be involved in the initiation of plus- strand RNA synthesis. Alternatively, inefficient plus-strand RNA synthesis in vitro would mimic the in vivo phenotype of this replicase (18) and may simply reflect the nature of a replication complex consisting of uncleaved P123 and nsP4. Processing of this complex in vitro may be necessary to convert it to a plus-strand replicase which is active enough to allow detection of plus-strand genomic and subgenomic RNA syn- thesis. It will be of interest to examine the polarity of RNA products synthesized with the nsP1 1 P23 1 nsP4 replicase, since this complex can synthesize plus-strand RNA in vivo (15). The SIN replicase exhibited specificity in that it did not utilize a number of RNA templates tested as substrates. The highest efficiency was observed for the SIN genome RNA, but the SIN replicase could also utilize Semliki Forest virus and Ross River virus RNAs. This is likely due to the conserved sequence elements that are found at the 59 and 39 ends of all alphavirus genomes that are believed to function as cis ele- ments for initiation of RNA synthesis. It has been postulated that the 19-base element adjacent to the poly(A) tail and se- quences in the 59 NTR or nsP coding region, in particular the 51-nucleotide element near the 59 end, may play a role in minus-strand initiation (37). Analyses of artificial 59 or 39 NTR chimeras between SIN and Ross River virus have demon-
Regulation of gene expression by transcriptional repression or activation has long been recognized as an effective means to control a biological process. However, while the initiation of an event can be governed by turning on the gene encoding a key rate-limiting enzyme mediating the respective action, its ter- mination must often be equally tightly controlled by inactivat- ing the responsible factor. Complete destruction is possibly the most effective way of ensuring the irreversible inactivation of a protein; consequently, all organisms employ intracellular pro- teolytic systems for the selective removal of “unwanted” pro- teins. This category includes short-lived regulatory factors as well as proteins that have been damaged or incapacitated by heat or other types of stress or toxic agents. In eukaryotes, regulated proteolysis is mediated largely by the 26S protea- some, a multicatalytic protease that consists of a barrel-shaped proteolytic 20S core particle in association with a 19S cap complex (20, 126). In contrast to a large portion of bulk protein turnover, which is mediated by vacuolar or lysosomal pro- teases, proteolysis by the 26S proteasome is energy dependent, due to the presence of ATPases of the AAA type within the 19S cap, which are responsible for unfolding the target pro- teins (135). Simpler versions of the 20S core and its associated ATPase subunits are known in archaebacteria and some eu- bacteria (76, 132). Even those bacteria that lack a conserved 20S particle employ a related strategy for energy-dependent proteolysis, using proteases that resemble the proteasome core particle architecturally and associate with a specific AAA ATPase subunit (6, 93, 132).
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Motifs I, II, and III were identified in rolling-circle initiator proteins nearly 20 years ago and subsequently shown to be essential for replication of many viral and plasmid DNAs (34, 40, 42, 52, 54). In the present study, we uncovered another sequence, the GRS, which displays strong conservation across all geminivirus Rep proteins (Fig. 1 and Fig. S1 in the supple- mental material). Its discovery was facilitated by the large number of geminiviruses genome sequences that have been reported in recent years (21). The GRS represents the longest consecutive stretch of near-amino-acid identity in the overlap- ping DNA binding and cleavage domains of the Rep protein. GRS mutants are not infectious, do not support viral genome replication, and are not competent for ssDNA cleavage, estab- lishing that the GRS is required for initiation of rolling-circle replication during geminivirus infection. GRS-related se- quences also occur in the Rep proteins of phytoplasmal and algal plasmids but not those of nanoviruses and circoviruses. Together, the phylogenetic and functional data established that the GRS is a conserved, essential motif characteristic of an ancient lineage of rolling-circle initiators and supported the idea that geminiviruses evolved from plasmids associated with phytoplasma or algae (41, 49, 58).
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The only other nonsegmented negative-strand RNA vi- ruses possessing bipartite promoters belong to the subfamily Paramyxovirinae (17, 18, 23, 30, 39, 44, 45). In contrast to filoviruses, the members of the Paramyxovirinae subfamily obey the rule of six, i.e., the total genome length must be a multiple of six to be efficiently replicated and transcribed (4, 8, 21, 33, 36). The overall replication promoter structure of the viruses in the Paramyxovirinae subfamily is similar. The first promoter element is located within the leader region and spans the first 12 to 36 nt of the genome. As with the filoviruses, the tran- scription start signal of the first gene does not belong to the replication promoter but is part of the spacer region. The second promoter element of most members of the Paramyxo- virinae consists of a stretch of three consecutive hexamers containing conserved C or CG residues (18, 23, 30, 39, 44, 45). Despite the hexameric phasing of the second promoter ele- ment of MARV and ZEBOV, filovirus genomes are not a multiple of six or another common integer (3). The lack of an integer length rule is one of the features filoviruses share with the members of the Pneumovirinae subfamily, such as hRSV. There are other similarities between filoviruses and pneumovi- ruses, e.g., the possession of a fourth nucleocapsid protein, which is unique among the nonsegmented negative-strand RNA viruses (1, 5). In contrast to the filoviruses, however, the replication FIG. 8. Comparison of the genomic replication promoter of ZEBOV and MARV. Promoter elements (PE) for replication are represented by black boxes, the transcription start signal (TSS) of the NP gene is shown in gray, and additional sequences belonging to the spacer region are indicated in a lighter gray. Regions of unimportant sequence are shown with hatching. Nucleotides involved in secondary structure formation are boxed in the sequence below the scheme. Right insets, scheme of predicted RNA secondary structures of these regions. The NP transcription start signal is indicated by a solid line in the insets and is underlined in the sequence. (A) The ZEBOV replication promoter is bipartite (PE1 and PE2). The spacer region includes the transcription start signal as well as the downstream sequence involved in secondary structure formation and can be extended or reduced by a multiple of 6 nt. PE2 consists of eight hexamers with U residues (boldface) at positions 81, 87,…123. (B) 3 ⬘ end of the MARV genome. PE2 of the MARV replication promoter is shorter, containing 3 UN 5 hexamers. The spacer region between PE1 and PE2
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palindrome do not abolish replication. The promiscuity of E1 in replication has also been demonstrated in a cell-free system in which origin-independent replication occurs at high concen- trations of BPV-1 E1 protein and in the ability of BPV-1 E1 protein to initiate unwinding in an origin-independent manner (87). There is considerable evidence that E1 and E2 interact to form a complex on DNA and that in so doing, E2 can increase the affinity of E1 binding to its recognition site (4, 50, 58, 67, 86, 88). BPV-1 E1 mutant proteins defective for origin binding can bind DNA specifically in the presence of E2 and can support replication from the BPV-1 origin in a cell culture system (76). Furthermore, mutated BPV-1 origins with dra- matically reduced capacity for E1 binding can nevertheless bind E1 in the presence of E2 (70). The implication is that the E1 helicase can be directed to DNA via interaction with the E2 protein bound at its recognition sites. Given that the E1 heli- case protein must bind DNA to catalyze unwinding, it will be interesting to determine exactly where the HPV-11 E1 protein binds a DNA template with a mutated E1BS in order to initiate replication. It is conceivable that E1 binds to sequences in the origin or plasmid which resemble the consensus E1BS, perhaps interacting with E2 over a distance. Certainly it has been shown that BPV-1 E2BSs can function in replication when placed at a distance from the E1BS, particularly if the E2BSs possess high affinity for E2 protein or are present in multiple copies (80). Alternatively, E1 may bind the DNA in a nonspecific manner, guided by its interaction with E2. It is likely that for HPV-11, as for BPV-1 (70, 86), tethering of E1 or the E1-E2 complex to the origin is necessary but not sufficient for repli- cation and therefore other cis-acting determinants, such as the AT domains and purine tracts mentioned, may allow for DNA conformation and unwinding suitability for initiation.
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DNA replication is a highly regulated cellular process in proliferating cells, involving cell cycle dependent assembly of DNA replication-initiation proteins (DRIPs) onto origins of replication. The process of pre-replicative complex (pre-RC) formation at the M-to-G 1 transition, also known as replication licensing, requires origin recognition complex (Orc1-6p) that binds and marks replication origins to facilitate the loading of additional DRIPs, such as Noc3p, Ipi3p, Cdc6p, Cdt1p and Mcm2-7p. The subsequent activation of pre-RC at the G 1 -to-S transition is dependent upon cyclin-dependent kinases (CDKs) and Dbf4-dependent kinase (DDK). This sequential process ensures that DRIPs are precisely loaded to form pre-ICs and then activated by their regulators so that chromosomal DNA is replicated only once per cell cycle. Despite substantial gains in the study of the mechanisms and regulation of pre-RC, the finite details of the pre-RC assembly and disassembly processes remain unclear and controversial. In this review we describe the present state of understanding on DRIPs and the pre-RC architecture and dynamics.
The data presented in chapter 3 of this study shows that pTP and poi of adenovirus type 5 individually and as a pTP-poi heterodimer bind with high specificity to the terminal 18bp of the adenovirus genome, forming nucieoprotein complexes which are readily detectable by gel retardation assay. This suggests that the mechanism by which pTP and poi, which exist In vivo as a tightly associated pTP-pol complex (Enomoto etal., 1981), locate themselves specifically at the origin prior to initiation of replication by recognition and binding to a sequence or sequences within the 1-18 domain. Functionally important domains of Ad poi have been identified through introduction of linker insertion mutations into the cloned Ad poi gene (Chen and Horwitz, 1989). Functional analyses on a range of such mutants showed that for the most part mutant proteins which were unabie to form a heterodimer with pTP also failed to recognise origin DNA. However one of the mutants studied was found to be abie
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When the ratio of fork pausing at each Ter site was investigated in the chromosomal context (i.e. fork pausing at a given Ter site depends on whether or not this site encounters a replication fork at the non-permissive face), TerE and TerJ were classified as pTer sites along with the last four Ter sites and TerF, based on the characteristic that no Y-shaped DNA intermediate was observed at these sites under endogenous Tus expression (Duggin and Bell, 2009). Using ChIP-qPCR, TerE was demonstrated to be a true Tus binding site that should be significantly occupied by Tus under natural conditions. Since TerE has been previously shown to be an efficient fork barrier (Duggin and Bell 2009, Esnault et al., 2007, Hidaka et al., 1991), it was concluded that TerE does not encounter replication forks moving towards its non-permissive face. This also suggested that TerH and TerI should not encounter oriC- initiated replication forks at their non-permissive face either, although some pausing was detected by Duggin and Bell (2009). The pausing at these outermost Ter sites observed by Duggin and Bell must therefore be the result of non-oriC initiated replication forks travelling towards the oriC. This is supported somewhat by the fact that pausing was increased at these sites upon Tus-overexpression which should result in a tighter inner fork trap and increased inhibition of fork progression towards the outermost Ter sites.
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