Premeiotic DNA replication

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Cdc28 and Ime2 Possess Redundant Functions in Promoting Entry Into Premeiotic DNA Replication in Saccharomyces cerevisiae

Cdc28 and Ime2 Possess Redundant Functions in Promoting Entry Into Premeiotic DNA Replication in Saccharomyces cerevisiae

Cdc28 is not required for premeiotic DNA replication and meiotic recombination: FACS analysis revealed that following 2 hr incubation at 37⬚ vegetative diploid cells homozygous for cdc28-deg arrested at G1, with a 2C DNA content (Figures 3A and 4B, time 0). These arrested cells were transferred to a sporulation medium and incu- bated at either 25⬚ or 34⬚. At both the permissive and nonpermissive temperatures, premeiotic DNA replica- tion took place (Figure 3A). Quantitative analysis of the results presented in Figure 3 revealed that in the ab- sence of any Cdc28 activity DNA replication was initiated at the same time (between 4 and 6 hr in SPM) at both the permissive and nonpermissive temperatures (Figure 4B). At 10 hr in SPM, 67% of the cells incubated at 25⬚ had a 4C DNA content, whereas at 34⬚, 39% had a 4C DNA content, reaching a maximum of 41.5% at 12 hr (Figure 4B). At 24 hr in SPM, at both temperatures, cells with 4C as well as with 2C DNA content were observed (Figure 3A), suggesting that DNA replication was either not complete or totally absent in a fraction of the cells. As a control, the same protocol was applied for the heterozygote isogenic strain. The 2-hr incubation at 37⬚ did not cause cell cycle arrest (Figure 3B). When these cells were shifted to SPM and incubated at 34⬚, cells completed the mitotic cycle, and, at 2 hr, most cells had Figure 3.—Cdc28 is not essential for premeiotic DNA repli- a 2C DNA content. At 4 hr, premeiotic DNA replication cation. Diploid cells homozygous for cdc28-deg (Y1314; A) or was initiated, and at ⵑ12 hr it was completed. Only a small heterozygous (Y1315; B) were grown at 25⬚ in PSP2 to a titer
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The Schizosaccharomyces pombe cdt2+ Gene, a Target of G1-S Phase-Specific Transcription Factor Complex DSC1, Is Required for Mitotic and Premeiotic DNA Replication

The Schizosaccharomyces pombe cdt2+ Gene, a Target of G1-S Phase-Specific Transcription Factor Complex DSC1, Is Required for Mitotic and Premeiotic DNA Replication

We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1 ⫹ /cdt2 ⫹ was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2 ⫹ was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2 ⫹ expression in vivo, and a cdt2 ⫹ promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30⬚ and were unable to grow at 19⬚. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline- 1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22 ⫹ , a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19⬚. These observations suggest that cdt2 ⫹ plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.
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Investigation of the Mechanism of Meiotic DNA Cleavage by VMA1-Derived Endonuclease Uncovers a Meiotic Alteration in Chromatin Structure around the Target Site

Investigation of the Mechanism of Meiotic DNA Cleavage by VMA1-Derived Endonuclease Uncovers a Meiotic Alteration in Chromatin Structure around the Target Site

To examine detailed requirements of meiotic progression for VDE-mediated DSBs, we introduced a mutation in genes of key regulators of meiotic events and observed homing by Southern analysis. In the ime1⌬ and ime2⌬ mutants, which are defective in the activation of early events of meiosis, including entry into the meiotic program and the induction of premeiotic DNA replication (6, 7, 37), VDE-mediated DSBs and homing products were not observed (Fig. 1C). In contrast to the case of the clb5 ⌬ clb6 ⌬ double mutant, in which the progression of premeiotic DNA replication is prevented (35), VDE-mediated DSBs were hardly observed at later time points in these mu- tants (Fig. 1C). These results suggest that entry into the mei- otic program is indispensable for the activation of VDE-me- diated DSB formation. As shown in Fig. 1D, VDE was localized both in the cytoplasm and in the nucleus in each mutant after 4 h of incubation in sporulation medium (SPM). Thus, it is suggested that a nutritional limitation only, which is sufficient for nuclear entry of VDE, is insufficient for the in- duction of homing, consistent with the results for NLS-fused VDE (Fig. 1B). On the contrary, the ndt80 ⌬ mutant, which leads to a failure to induce the middle sporulation genes and a subsequent arrest before nuclear divisions (4, 37), exhibited an induction of DSBs and intein-coding sequence homing (Fig. 1C), indicating that VDE-mediated DSB formation is an event which occurs before nuclear divisions. Thus, these results lead us to hypothesize that the nuclear-localized VDE, which was induced by nutritional limitation, is subjected to further regu- lation that inhibits VDE-mediated DSB formation until mei-
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Counteracting Regulation of Chromatin Remodeling at a Fission Yeast cAMP Responsive Element-Related Recombination Hotspot by Stress-Activated Protein Kinase, cAMP-Dependent Kinase and Meiosis Regulators

Counteracting Regulation of Chromatin Remodeling at a Fission Yeast cAMP Responsive Element-Related Recombination Hotspot by Stress-Activated Protein Kinase, cAMP-Dependent Kinase and Meiosis Regulators

In fission yeast, an ATF/CREB-family transcription factor Atf1-Pcr1 plays important roles in the activation of early meiotic processes via the stress-activated protein kinase (SAPK) and the cAMP-dependent protein kinase (PKA) pathways. In addition, Atf1-Pcr1 binds to a cAMP responsive element (CRE)-like sequence at the site of the ade6-M26 mutation, which results in local enhancement of meiotic recombination and chromatin remodeling. Here we studied the roles of meiosis-inducing signal transduction pathways in M26 chromatin remodeling. Chromatin analysis revealed that persistent activation of PKA in meiosis inhibited M26 chromatin remodeling, suggesting that the PKA pathway represses M26 chromatin remodel- ing. The SAPK pathway activated M26 chromatin remodeling, since mutants lacking a component of this pathway, the Wis1 or Spc1/Sty1 kinases, had no M26 chromatin remodeling. M26 chromatin remodeling also required the meiosis regulators Mei2 and Mei3 but not the subsequently acting regulators Sme2 and Mei4, suggesting that induction of M26 chromatin remodeling needs meiosis-inducing signals before premeiotic DNA replication. Similar meiotic chromatin remodeling occurred meiotically around natural M26 heptamer sequences. These results demonstrate the coordinated action of genetic and physiological factors required to remodel chromatin in preparation for high levels of meiotic recombination and eukaryotic cellular differentiation.
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Cyclin B-Cdk Activity Stimulates Meiotic Rereplication in Budding Yeast

Cyclin B-Cdk Activity Stimulates Meiotic Rereplication in Budding Yeast

Haploidization of gametes during meiosis requires a single round of premeiotic DNA replication (meiS) followed by two successive nuclear divisions. This study demonstrates that ectopic activation of cyclin B/cyclin-dependent kinase in budding yeast recruits up to 30% of meiotic cells to execute one to three additional rounds of meiS. Rereplication occurs prior to the meiotic nuclear divisions, indicating that this process is different from the postmeiotic mitoses observed in other fungi. The cells with overreplicated DNA produced asci containing up to 20 spores that were viable and haploid and demonstrated Mendelian marker segregation. Genetic tests indicated that these cells executed the meiosis I reductional division and possessed a spindle checkpoint. Finally, interfering with normal synaptonemal complex formation or recombination increased the efficiency of rereplication. These studies indicate that the block to rereplica- tion is very different in meiotic and mitotic cells and suggest a negative role for the recombination machinery in allowing rereplication. Moreover, the production of haploids, regardless of the genome content, suggests that the cell counts replication cycles, not chromosomes, in determining the number of nuclear divisions to execute.
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Virtual reality tool for teachers

Virtual reality tool for teachers

The first could-have was implemented with the ability for students to click on certain parts of the DNA and see what the name of the part is. However, this feature, as described in the specifications, has not fully been implemented. The different parts that should have been clickable had to include the sugar, phosphate, adenine, guanine, thymine, cytosine and the freely floating nucleotide inside the cell nucleus, as described in the specification. In like manner, this has been the case for at least one of each of these parts. Unfortunately, not all of the parts were clickable. The reason for this was that there was simply not enough time to make all the different parts clickable since there were a lot of parts and this would have taken a lot of time. For that reason, only a few that were easily reachable for the students were clickable. Apart from the MoSCoW analysis, the teacher had another requirement, an animation should be available to show how the process of separation of two DNA strings is visualized. The animation was also implemented in the lesson. However, in the specification chapter, another ‘animation’ was mentioned, namely the animation of going from the skin to the cell nucleus. This was not achieved since there was limited time and implementing this feature would mean a 3D model of skin on a human would have had to be made, for which there was no time left.
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A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome.

A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome.

We show here that a subset of HSV replication genes not sufficient for ori-dependent HSV DNA replication is necessary and sufficient for the induction of SV40 DNA amplification upon tran[r]

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Telomere Length Differences upon Keratinization and its Variations in Normal Human Epidermal Keratinocytes

Telomere Length Differences upon Keratinization and its Variations in Normal Human Epidermal Keratinocytes

keratinocytes with one very long telomere in each nucleus occasionally and independently exist in the epidermis. The modified end-replication problem, based on the 3’-overhanged DNA end structure, predicts that leading ends become shorter and lagging ends avoid telomere shortening during DNA replication [26]. As more cells divide, preserved telomeres would become less in frequency, but more conspicuous in length. We postulated that the reason for significant differences in telomere length among cell groups was as follows: 1) CPCs may give rise to daughter cells for comparatively long periods, leading to variations in telomere length between cell groups; and 2) CPCs derived from distinct SCs may be arranged side-by-side.
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Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

Prelabeled, depleted nuclei were incubated at two concentrations of nuclei with increasing amounts of cytoplasmic protein extracts from polyoma-infected cells microliters.. Incubations w[r]

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Cellular Topoisomerase I Modulates Origin Binding by Bovine Papillomavirus Type 1 E1

Cellular Topoisomerase I Modulates Origin Binding by Bovine Papillomavirus Type 1 E1

purified E1 in the presence and absence of Topo I. The pro- tein-DNA complexes were cross-linked with glutaraldehyde and subjected to gel electrophoresis. The binding of E1 to origin DNA resulted in an E1-DNA complex (Fig. 1A, lane 8). The addition of increasing amounts of Topo I to this complex resulted in increased binding of E1 to origin DNA. At higher levels of Topo I, a Topo I-DNA complex that migrated slightly faster than the E1-DNA complex was seen (Fig. 1A, Topo I, lanes 4 to 7 and 11 to 14). Quantification demonstrated the Topo I stimulation of origin binding activity by E1 to range from three- to sevenfold (Fig. 1B; also see Fig. 3C). To verify FIG. 1. Topo I stimulates the origin binding activity of E1. (A) A fixed amount of E1 (0.5 pmol) and increasing amounts of Topo I (0 to 200 fmol) were incubated with radiolabeled BPV1 origin DNA for 30 min at 37°C, followed by cross-linking with glutaraldehyde. The result- ing protein-DNA complexes were resolved using 5% polyacrylamide gel electrophoresis and analyzed using autoradiography. Migrations of the E1-DNA complex, the Topo I-DNA complex, and the free probe are indicated by arrowheads. (B) Results from panel A were subjected to phosphorimager analysis and quantified using Quantity One soft- ware. The radioactivity of the E1-DNA complex from each lane was quantified and compared to the radioactivity of the E1-DNA complex in the absence of Topo I (lane 8), which was assigned a value of 1. The results represent three separate experiments; the range is depicted by error bars.
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DNA METABOLISM DURING PACHYTENE IN RELATION TO CROSSING OVER

DNA METABOLISM DURING PACHYTENE IN RELATION TO CROSSING OVER

Analyses of DNA synthesis in the diploid hybrid show that normal DNA replication occurs during the premeiotic S phase and during zygotene but only a little synthesis can be detec[r]

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A Review of DNA Replication Studies and Contemporary Researches in Yeast

A Review of DNA Replication Studies and Contemporary Researches in Yeast

which depend upon the presence, absence or location of the origin within the restriction fragment. Thus the ‘bubble’ shaped RIs formed due to the presence of internal origins are separated from the ‘Y’ shaped RIs formed due to the absence of internal origins causing replication of the fragments by forks coming from external origins. These fragments can be seen after southern hybridization using suitable radioactive probes (Brewer and Fangman 1987). The second dimension electrophoresis of the neutral/alkaline technique (Huberman et al., 1987; Nawotka and Huberman 1988) is run under denaturing conditions so that the nascent strands of different sizes are fractionated. The restriction fragments generated from replicating DNA contain short nascent strands and the length of these short fragments at any point within the restriction fragment shows the distance of that point from its replication start point/initiation site. This technique is capable of measuring the sizes of various nascent strands generated by replicating restriction fragments and thus, can help to identify the direction of DNA replication and subsequently, the point from where the bi-directional replication starts – the origin. Soon after the pioneering origin mapping studies in ARS1 and yeast 2µ plasmids (Brewer and Fangman 1987; Huberman et al., 1987), a chromosomal origin was located near ARS305 of yeast chromosome III using the N/A 2D technique (Huberman et al., 1988). The subsequent years witnessed a boom in chromosomal origin mapping studies in yeast and other organisms. The rDNA repeats of chromosome XII (Linskens and Huberman 1988) and the three ARS elements on the left arm of yeast chromosome III, ARS306 (Deshpande and Nwelon 1992, Zhu et al., 1992a), ARS307 (Greenfeder and Newlon 1992) and ARS309 (Greenfeder and Newlon 1992) are some of the examples of the earliest mapped chromosomal origins using the 2D techniques. Although the replication origins were mapped at or near the ARS elements using 2D techniques, it was not clear whether the ARS sequences were sufficient for the origin function in the chromosome or some other factors were responsible for their function. Deshpande and Newlon (1992) deleted the chromosomal copy of ARS306 from chromosome III and replaced ARS307 with a mutated copy of ARS307 containing a mutation in the essential domain A. 2D gel analysis of the relevant regions showed abolition of origin activity suggesting that the chromosomal origin function was a property of ARS elements themselves.
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Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

The bound proteins eluted from the beads with BC500 or BC1000 buffer were also denatured in 2 ⫻ SDS in protein sample buffer containing 5% 2-mercaptoethanol by heating at 90°C for 5 min and were separated in a NuPAGE 4% to 12% bis-Tris gel in 1 ⫻ NuPAGE MES (morpholineethanesulfonic acid) SDS running buffer (Invitrogen). After being transferred onto a nitrocellulose membrane, the membrane was blocked with 5% nonfat milk–Tris-buffered saline (TBS) for 1 h at room temperature, rinsed with TBS, and incubated overnight at 4°C with a primary antibody. Subsequently, the membrane was washed 3 times with TTBS (TBS with the addition of Tween 20) at a final concentration of 0.1% (vol/vol) and incubated with a horseradish peroxidase-labeled secondary antibody (Sigma) diluted 5,000-fold in TTBS for 1 h at room temperature. After 3 thorough washes with TTBS, the immunoreactive proteins on the membrane were detected with enhanced chemiluminescence substrate (Pierce, Rockford, IL). The signal was captured on X-ray film. Before reprobing with another primary antibody was performed, the membrane was stripped with Restore Western blot stripping buffer (Pierce) according to the manufacturer’s instructions and blocked with 5% nonfat milk–TBS. The primary antibodies used were goat polyclonal anti-hnRNP D0 (T-10) (Santa Cruz) (1:100); mouse monoclonal anti-hnRNP A/B (G-10) (Santa Cruz) (1:100); mouse polyclonal anti-DNA polymerase beta (Abcam, Inc.; catalog no. 2856) (1:500); and anti- ␤ -tubulin (tub 2.1) (Sigma) (1:3,000).
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Examining a DNA Replication Requirement for Bacteriophage λ Red  and Rac Prophage RecET Promoted Recombination in Escherichia coli

Examining a DNA Replication Requirement for Bacteriophage λ Red and Rac Prophage RecET Promoted Recombination in Escherichia coli

The linear dimer plasmid substrate is a nonreplicating sub- strate, yet both the Red and RecET systems recombine it to form circular monomers proficiently. Taken together with the results of our other experiments using an oligonucleotide to target a non- replicating circular plasmid, these results demonstrate that the DNA replication requirement proposed for Red-mediated recom- bineering (33, 36, 37) depends on the DNA substrate and applies only to circular DNA molecules. Our experiments are consistent with both the Red and RecET systems acting predominately by single-strand annealing. The RecT-mediated oligonucleotide re- combination frequency on freely replicating plasmids is less ro- bust than that mediated by Beta, suggesting that RecT recombina- tion occurs less often at single-stranded gaps present at the DNA replication fork then does Red recombination. To explain this difference, we suggest that the Beta protein may be better able to displace the single-strand binding (SSB) protein from a DNA rep- lication fork. While we cannot rigorously rule out a low level of strand invasion in our circular-plasmid– oligonucleotide crosses, any strand invasion must be barely above the level of the back- ground recombination occurring in the absence of recombinases.
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DNA Replication in Plants Origins of DNA Replication and Post-translational Histone Modifications During the DNA Synthesis Phase of Arabidopsis thaliana.

DNA Replication in Plants Origins of DNA Replication and Post-translational Histone Modifications During the DNA Synthesis Phase of Arabidopsis thaliana.

The λ-exonuclease SNS procedure omits the use of BrdU and relies on an RNA primer to protect nascent DNA from the 5’ to 3’ exonuclease that is used to remove nicked DNA. Therefore, care must be taken to avoid RNases to keep RNA primer cap intact and protect the nascent strands from λ-exonuclease digestion. Another important consideration for the λ-exonuclease procedure is complete digestion of the unprotected DNA. If the unprotected DNA is not completely removed, it will cause a false signal when the final mapping analysis is done. Recently Cayrou, et al (2011) first size selected nascent strands population that was then digested with a specially prepared λ-exonuclease to thoroughly remove the contaminating, unprotected DNA to map the origins of DNA replication in Drosophila and mouse cells (Cayrou et al., 2011). The Cayrou, et al (2011) study has caused controversy because the results raise the possibility that earlier studies that relied on a different source of λ-exonuclease may have been subject to a high level of contamination from undigested DNA, which would have impacted the results of these studies. In addition, Cayrou, et al (2011) showed that the amount of small nascent strand DNA that remains following digestion with the specially prepared λ -exonuclease is vanishingly small (<20 ng per 10 8 nuclei), demonstrating the challenges faced by researchers who use this method.
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The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells

The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells

to cellular dsDNA when expressed even in the absence of viral DNA is not surprising, since ppUL44 appears to be able to bind to dsDNA without any sequence specificity (22). Underlining the physiological significance of the results, GFP- UL44 ⌬ loop failed to support HCMV oriLyt-dependent DNA replication in cells (Fig. 6). Similarly, mutations preventing ppUL44 homodimerization in a cellular context also prevented ppUL44 from transcomplementing viral DNA replication in a transient-transfection assay (41), most likely due to the inabil- ity of these mutants to bind to dsDNA (see Fig. 3 and 4). Although the experiments here were not performed using the preferred experimental system of recombinant viruses, the re- sults obtained are clearly consistent with a recent report show- ing that mutations impairing HSV-1 UL42 ability to bind to dsDNA also impair replication of a recombinant virus (20) and thus strong evidence for an important role for ppUL44-FL in HCMV replication. Importantly, the defect of ppUL44 ⌬ loop in mediating oriLyt-dependent DNA replication is not due to misfolding of the protein, as indicated by its ability to both bind to pUL54 and to heterodimerize with ppUL44wt (see Fig. 5AB and see Fig. S2 in the supplemental material). Hence, our results suggest that the inability of ppUL44 ⌬ loop to transcomplement oriLyt-dependent DNA replication depends directly on its re-
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Interactions of the Papovavirus DNA Replication Initiator Proteins, Bovine Papillomavirus Type 1 E1 and Simian Virus 40 Large T Antigen, with Human Replication Protein A

Interactions of the Papovavirus DNA Replication Initiator Proteins, Bovine Papillomavirus Type 1 E1 and Simian Virus 40 Large T Antigen, with Human Replication Protein A

It has been shown that the RPA32 and RPA14 homologs are essential in S. cerevisiae, and mutants exhibit phenotypes con- sistent with a block in DNA replication (7, 30). Furthermore, antibodies against RPA32 subunits have been shown to inhibit SV40 DNA replication in vitro (33). Therefore, it was surpris- ing that high levels of MBP-RPA14 or MBP-RPA32 did not inhibit SV40 DNA replication in vitro. One possible explana- tion is that the MBP domain is precluding these proteins from interacting with their natural partners, so that the fusion pro- teins do not compete with the RPA complex for essential interactions. Alternatively, the DNA replication functions of RPA14 and RPA32 may be merely to stabilize the RPA70 subunit, as previously suggested (74). While RPA14 and RPA32 may have additional functions (in DNA repair or re- combination, for example), their requirement to form the het- erotrimer so that RPA70 can support DNA replication may complicate the analysis of any other roles the smaller subunits may play. The ability of antibodies against RPA32 to inhibit SV40 DNA replication may be due to steric limitations at a very crowded DNA replication fork. It is still unclear whether RPA32 and RPA14 play a direct, vital role in DNA replication. We propose that the polyomaviruses and the papillomavi- ruses have evolved similar mechanisms to appropriate the cel- lular DNA replication machinery. Both types of viruses have evolved a protein that recognizes the viral origin, acts as a DNA helicase, and binds to the two cellular DNA replication complexes, RPA and DNA polymerase a-primase. We pro- pose that as these two cellular complexes are recruited to the viral DNA replication fork, they in turn recruit the other re- quired cellular DNA replication proteins.
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ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication

ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication

Notably, this is the first study that identifies a ‘reader’ of newly synthesized histone H4 di-acetylation marks at K5 and K12. Although this histone modifications are evolutionally conserved from yeast to mammals and known for past decades [5, 26], their function during DNA replication remained largely enigmatic. Our results lead to a new hypothesis how ATAD2 and new histone marks could work in DNA replication. They might regulate the kinetics of nascent chromatin maturation. Higher order chromatin structure formation requires tight regulation of acetylation and deacetylation [27]. Our results suggest that ATAD2 may regulate deacetylation of new histone marks by competing with HDAC1, perhaps to assist proper heterochromatin compaction. ATAD2 was indeed found enriched at heterochromatin replication foci and associated with the heterochromatin component (Figure 8). Previous studies support this possibility. New histone marks have been shown to be more strongly associated with heterochromatin duplication [28] and manipulation of ATAD2 expression levels resulted in altered chromatin compaction in S. cerevisiae as well as in embryonic stem cells [29, 30]. Hence, it is tempting to speculate that ATAD2 is initially recruited to nascent chromatin through its interaction with new histone marks and then assists heterochromatin compaction by regulating the acetylation status of new histones.
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Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen.

Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen.

Although we already knew that the SV2905 genome was defective for viral DNA replication in vivo, we tested the purified 2905T in an SV40 DNA replication assay in vitro 48.. Our assay was[r]

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Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle.

Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle.

Human papillomaviruses (HPVs) are small, double- stranded DNA viruses that infect epithelial cells. Over 70 dif- ferent HPV genotypes have been identified. A subset of HPVs primarily cause anogenital warts and have been placed into two groups, low risk and high risk. The anogenital HPVs in the low-risk group lead only to the production of benign lesions or warts, while HPVs in the high-risk group, HPV type 16 (HPV- 16), HPV-18, HPV-31, and HPV-33, are associated with 90% of cervical cancers (36). HPV-16 is the most common subtype associated with cervical cancers. The papillomaviral life cycle is tied to the differentiation of its host, epithelial cells. The virus is thought to gain access to the basal epithelial cells at a site of wounding. The life cycle can be separated into two stages, nonproductive and productive. In the nonproductive stage, the viral genome is established as a low-copy-number nuclear plas- mid. This occurs in the proliferating basal layer of the epithe- lium, where the virus replicates its DNA to keep up with the division of basal and parabasal cells and establishes a steady- state level of viral genomes (6). As these cells undergo their normal life cycle, a subset of daughter cells become detached from the basement membrane, stratify, and differentiate. The productive stage of the viral life cycle occurs in the terminally differentiating layers of the epithelium. In these cells, the virus amplifies its genome to higher copy number, expresses late genes encoding the capsid proteins, and produces viral prog- eny.
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