DNA replication and repair

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Mismatch Repair of DNA Replication Errors Contributes to Microevolution in the Pathogenic Fungus Cryptococcus neoformans

Mismatch Repair of DNA Replication Errors Contributes to Microevolution in the Pathogenic Fungus Cryptococcus neoformans

Microevolution in fungal pathogens has previously been attributed to changes at the chromosomal level, with evidence of gain, loss, or recombination between chro- mosomes leading to resistance to antifungal agents (1–5). Upon exposure to the antifungal agent fluconazole, Candida albicans and Cryptococcus neoformans undergo a process termed heteroresistance in which rapid and yet reversible resistance is conferred by the development of one or more aneuploidies or large-scale chromosomal rearrangements (reviewed in reference 6). The most commonly occurring aneuploidy in C. albicans is an isochromosome of the left arm of chromosome 5, i(5L), which contains TAC1 and ERG11, genes encoding the transcriptional activator of drug efflux genes and the target of fluconazole (7, 8). In C. neoformans, increasing concentrations of flucona- zole result in duplication of chromosome 1, containing ERG11 and the azole transporter AFR1, in all resistant strains, followed by successive duplication of chromosomes 4, 10, and 14 (9, 10). Cells return to normal ploidy when fluconazole is removed, presumably due to reduced fitness as evidenced by reduced proliferation and virulence in vivo (10). Interestingly, recent studies have also indicated that single-base-pair mutations may play an important role in the microevolution of pathogenic fungi during infection (11, 12). Genomic sequencing of C. neoformans isolates from a relapsed patient before and after antifungal treatment identified a chromosomal rearrangement and also an addi- tional base pair mutation in the AVC1 gene that controls several virulence phenotypes (11). In addition, high-frequency switching of C. neoformans between yeast cells to a pseudohyphal cellular morphology is governed by single-base-pair mutations in genes of the regulation of Ace2p activity and cellular morphogenesis (RAM) pathway (12). Importantly, mutation of MSH2, MLH1, and PMS1 (three C. neoformans genes from the mismatch repair [MMR] pathway predicted to be required to repair damage to single bases arising from errors in DNA replication) resulted in increased proliferation in a lung assay of cryptococcosis (13). This suggests that single-base mutations arising from errors in DNA replication can generate novel traits that facilitate the ability to cause disease and therefore provide an avenue for the pathogen to undergo microevolution during infection.

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DNA replication roadblocks caused by Cascade Interference complexes are alleviated by RecG DNA repair helicase

DNA replication roadblocks caused by Cascade Interference complexes are alleviated by RecG DNA repair helicase

CRISPR-Cas immunity therefore relies on insertion of spacer DNA into CRISPR loci. This occurs by processes collectively called ‘ Adaptation ’ that involve the capture of MGE DNA fragments and their subsequent integration into a CRISPR locus. Adaptation in E. coli is dependent on Cas1 and Cas2 proteins forming an oligomer of two Cas1 dimers held together by a Cas2 dimer [2,3]. This forms a pre-integra- tion complex with a flayed duplex DNA molecule that posi- tions DNA 3´ OH groups appropriately for integration into CRISPR as a new spacer [4,5]. Integration occurs via transes- terification reactions that have been elucidated in detail [6–9]. Adaptation events prior to DNA integration require DNA pre-processing into molecules suitable for capture by Cas1- Cas2. It is not clear how this occurs but genetic analysis has implicated various host cell nucleases and helicases including involvement of enzymes from host DNA replication and DNA repair pathways [10–12]. The genetic requirements for adap- tation also vary according to whether Cas1-Cas2 is establish- ing new immunity when there is no interference because an

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DNA replication roadblocks caused by Cascade Interference complexes are alleviated by RecG DNA repair helicase.

DNA replication roadblocks caused by Cascade Interference complexes are alleviated by RecG DNA repair helicase.

CRISPR-Cas immunity therefore relies on insertion of spacer DNA into CRISPR loci. This occurs by processes collectively called ‘ Adaptation ’ that involve the capture of MGE DNA fragments and their subsequent integration into a CRISPR locus. Adaptation in E. coli is dependent on Cas1 and Cas2 proteins forming an oligomer of two Cas1 dimers held together by a Cas2 dimer [2,3]. This forms a pre-integra- tion complex with a flayed duplex DNA molecule that posi- tions DNA 3´ OH groups appropriately for integration into CRISPR as a new spacer [4,5]. Integration occurs via transes- terification reactions that have been elucidated in detail [6–9]. Adaptation events prior to DNA integration require DNA pre-processing into molecules suitable for capture by Cas1- Cas2. It is not clear how this occurs but genetic analysis has implicated various host cell nucleases and helicases including involvement of enzymes from host DNA replication and DNA repair pathways [10–12]. The genetic requirements for adap- tation also vary according to whether Cas1-Cas2 is establish- ing new immunity when there is no interference because an

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DNA replication roadblocks caused by Cascade Interference complexes are alleviated by RecG DNA repair helicase.

DNA replication roadblocks caused by Cascade Interference complexes are alleviated by RecG DNA repair helicase.

dimers held together by a Cas2 dimer (Nunez et al. 2014; Nunez et al. 2015a). This forms a pre-integration complex with a flayed duplex DNA molecule that positions DNA 3« OH groups appropriately for integration into CRISPR as a new spacer (Nunez et al. 2015b; Wang et al. 2015). Integration occurs via transesterification reactions that have been elucidated in detail (Nunez et al. 2016; Pougach et al. 2010; Rollie et al. 2015; Wright et al. 2017). Adaptation events prior to DNA integration require DNA pre- processing into molecules suitable for capture by Cas1-Cas2. It is not clear how this occurs but genetic analysis has implicated various host cell nucleases and helicases including involvement of enzymes from host DNA replication and DNA repair pathways (Ivancic-Bace et al. 2015; Kunne et al. 2016; Levy et al. 2015). The genetic requirements for adaptation also vary according to whether Cas1-Cas2 is establishing new immunity when there is no interference because an MGE has not been previously encountered (“naïve” adap tation), or if Cas1-Cas2 is updating immunity after interference has recognized an MGE (“targeted/primed” adaptation).

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The role of ChlR1 in DNA replication, DNA damage repair and cohesion establishment

The role of ChlR1 in DNA replication, DNA damage repair and cohesion establishment

The results obtained in this thesis suggesting a role for ChlR1 in DNA replication, are in argeement with results from previous studies. ChlR1 interacts with a number of proteins involved in DNA replication including Mcm7, PCNA, TopBP1 (Feeney and Parish, unpublished) and Fen1 [50]. Furthermore ChlR1 stimulates the endonuclease in vitro activity of Fen1 three fold [50]. In addition, a number of other cohesion establishment factors associate with the replication fork, including Ctf18 and Esco1 and 2. A number of related helicases are involved in DNA replication. FANCJ has been shown to function in DNA replication checkpoint control [311]. FANCJ interacts with TopBP1 and FANCJ knockdown results in the inability of RPA to load onto the DNA [311]. This is a prerequisite for ATR checkpoint activation. FANCJ is involved in the phosphorylation of both Chk1 and RPA following replication stress [311]. These results suggest FANCJ has a role in the restarting of stalled replication forks after DNA damage. RecF is another related helicase involved in DNA replication. In E.coli depleted of RecF replication fails to recover after UV damage [320]. Furthermore RecF is important in protecting the nascent DNA at stalled replication forks [321]. In the absence of RecF in E coli the nascent strand is degraded at these stalled replication forks [321].

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Characterization of the Repeat-Tract Instability and Mutator Phenotypes Conferred by a Tn3 Insertion in RFC1, the Large Subunit of the Yeast Clamp Loader

Characterization of the Repeat-Tract Instability and Mutator Phenotypes Conferred by a Tn3 Insertion in RFC1, the Large Subunit of the Yeast Clamp Loader

this idea; moreover, pol30-52 is defective in homotri- A similar relationship between mismatch repair and meric interactions and is also defective in in vitro DNA RFC functions was not observed in the repeat-tract insta- replication reactions (Table 3; Ayyagari et al. 1995). bility assay because the rfc1::Tn3 msh2 or rfc1::Tn3 pms1 Further support is provided from the work of Kokoska double mutants displayed a mutator phenotype that was et al. (1999, accompanying article) in which they ana- indistinguishable from that observed in msh2 or pms1 lyzed the effect of the pol30-52 mutation on micro- and single mutants. The different phenotypes in these two minisatellite instability and found that, in addition to assays were not unexpected considering that RFC1, conferring a defect in mismatch repair, the pol30-52 MSH2, and PMS1 are likely to be functioning in multi- mutation conferred a minisatellite instability pheno- subunit DNA replication and repair machines and it is type. They hypothesized that this phenotype was the difficult to determine which effects are direct and which result of the pol30-52 mutation affecting DNA replica- are indirect. Three possible explanations for these dif- tion by increasing the rate of DNA polymerase slippage. ferences are as follows:

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Redox Signaling in Eukaryotic DNA Replication and Repair

Redox Signaling in Eukaryotic DNA Replication and Repair

To characterize initiation, we used a 50-nt ssDNA substrate (Table 2.1) containing exactly one complementary base for the radiolabeled nucleotide triphosphate, α- 32 P ATP. We found that WT primase has much higher overall initiation activity than both mutants tested, including the single-atom p48/p58Y345F variant (Figure 2.15A, 2.15B). CT-deficient primase mutants are significantly less active on ssDNA, synthesizing only 15-35% of the WT initiation products over multiple trials. Compared to WT primase, a larger portion of the products synthesized by the mutants is < 10 nucleotides in length, suggesting that the rate-determining initiation step in product synthesis is inhibited for mutants deficient in the redox switch. The significant difference in initiation activity for the WT and CT-deficient mutant primase enzymes suggests that a redox switch changing the oxidation state of the [4Fe4S] cluster in p58C is crucial for the reaction to begin primer synthesis. Additionally, the difference in activity on ssDNA caused by a very subtle mutation in the CT pathway suggests that charge migration from bound DNA to the [4Fe4S] cofactor, through the insulating protein matrix of p58C, plays a significant role in mediating the rate-determining primase activation step on genomic template DNA, prior to the presence of duplex RNA/DNA.

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Elucidating the Role of [4Fe4S] Clusters in DNA Replication and Repair Proteins

Elucidating the Role of [4Fe4S] Clusters in DNA Replication and Repair Proteins

reproducible, and a potential shift upon DNA binding is observed. With respect to studying proteins in the absence of DNA, the PGE electrode represents a significant advance over previously used highly-oriented pyrolytic graphite (HOPG), which is hydrophobic and difficult to prepare. To test the effect of protein environment on redox potential, a series of EndoIII point mutants were prepared in which the charge within 5 Å of the cluster was reversed or added in. None of these mutations induced a significant shift in redox potential relative to wild type, arguing that DNA electrostatics are the dominant factor in potential modulation. In parallel, XAS studies were performed on EndoIII and MutY in the presence and absence of DNA, and in the presence and absence of solvent. Ligating cysteinyl thiols and inorganic S atoms in the [4Fe4S] cluster absorb at different intensities in XAS depending on solvent environment and local electrostatics; these changes, in turn, directly correlate to redox potential. By XAS, DNA was found to induce a significant shift in absorbance, and thus potential; the removal of solvent had a smaller effect. Together, these studies provide new approaches for the study of DNA-binding [4Fe4S] proteins and reveal the critical role of DNA in tuning the redox potential.

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Molecular pathogenesis in granulosa cell tumor is not only due to somatic FOXL2 mutation

Molecular pathogenesis in granulosa cell tumor is not only due to somatic FOXL2 mutation

heterozygous FOXL2 402C > G mutation in the tumor by direct gene sequencing. In addition, DNA replication error, on analysis of the lengths of CAG repeats in androgen receptor gene, revealed defective DNA mismatch repair system in the granulosa cell tumor. We propose that the 402C > G mutation in FOXL2 is critical to the development of adult granulosa cell tumor. However, the malignant behavior of this tumor is driven by DNA mismatch repair deficiency. Unequal DNA copy numbers were noted on array comparative genomic hybridization. This implies that there is malignant potential even in the early stage of the granulosa cell tumor. Late malignant recurrence may be a late event of DNA repair function disability, not directly related to pathognomonic FOXL2 mutation.

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<p>CDKN3 promotes tumor progression and confers cisplatin resistance via RAD51 in esophageal cancer</p>

<p>CDKN3 promotes tumor progression and confers cisplatin resistance via RAD51 in esophageal cancer</p>

In the pilot study, we demonstrated that the CDKN3 expression was signi fi cantly increased in both esophageal adenocarcinoma (EA) and esophageal squamous cell car- cinoma (ESCC) in comparison with normal esophageal epithelial tissues. Elevated levels of CDKN3 is associated with a poor prognosis in ESCA. Interestingly, bioinfor- matic analysis of The Cancer Genome Atlas (TCGA) database revealed CDKN3 involvement in numerous bio- logical processes including cell cycle transformation, DNA replication and DNA damage repair (DDR), et al Therefore, we hypothesized that CDKN3 promotes tumor progression through cell cycle regulation and contributes to chemoresistance through DDR. We also found that CDKN3 expression was positively correlated with RAD51 expression, which mediates homologous pairing and strand exchange reactions between single and double stranded DNA during repair. 7 In all, we determined that CDKN3 can promote cell proliferation by facilitating the cell cycle G2/M transition and in fl uencing chemosensitiv- ity by interacting with RAD51 in ESCA.

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Characterisation of non essential genes required for survival under conditions of DNA stress

Characterisation of non essential genes required for survival under conditions of DNA stress

The aim of this experiment was to observe and compare the DNA stalled replication fork profiles, in terms of accumulation and resolution of DNA forks, of a rad4-116 strain to hip1Δ and wildtype rad4 as a control. This ability to assess and monitor such profile would be achieved by utilising Rad52-GFP which is recruited to repair and aid the progress of stalled replication forks. The strains were constructed by direct transformation of PCR-produced deletion hip1 encoding G418 resistance and PCR-produced rad4-116 attached to NAT resistance into a rad52-GFP base strain. Cells were grown in liquid media then arrested by the addition of hydroxyurea which blocks ribonucleotide reductase leading to the depletion of the ribonucleotides, thus halting DNA replication. Then after 6 hours of arrest with hydroxyurea, the cells were released by the depletion of hydorxyurea from the media, washing the cells then resuspending in rich YES media. Samples were collected every 30 minutes for 210 minutes to be analysed for fluorescence microscopy and testing viability.

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DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells

DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells

S-phase factors and DNA repair DNA polymerases are recruited into centers of viral DNA replication. Since S-phase arrest was not observed in cells replicating the HBoV1 DNA (Fig. 10), we investigated the role of the S phase in the replication of HBoV1 DNA. We examined the localization of key S-phase (DNA replication) factors, including proliferating cell nuclear antigen (PCNA), replication factor C1 (RFC1), polymerase ␣ (Pol FIG 8 Inhibition of phosphorylation of ATM, ATR, or DNA-PKcs significantly impairs replication of the HBoV1 DNA. HEK293 cells were treated with DMSO or the indicated inhibitor for 4 h prior to transfection with pIHBoV1. At 48 h posttransfection, cells were collected for Southern and Western blot analyses. HU-treated cells were used as a positive control for DDR induction. (A and B) Southern blotting. (A) Representative Southern blot of Hirt extracts probed for HBoV1 NS and Cap. The detected bands are labeled as follows: dRF, double replicative form; mRF, monomer replicative form; and ssDNA, single-stranded DNA. (B) Quantification of mRF DNA in the blot following normalization to the amount of DpnI-digested DNA. The data are based on the results of three independent experiments. Means and standard deviations are shown. (C) Quantification of progeny virus produced. Virus was purified from HEK293 cells transfected with pIHBoV1 for 48 h. Shown are the average numbers of vgc of purified progeny virions per preparation and standard deviations. (D and E) Western blot analysis. (D) Cell lysates were blotted using anti-p-RPA32. The blot was reprobed with anti- ␥ H2AX and ␤ -actin, in that order. (E) Cell lysates were blotted using anti-p-ATM(S1981), anti-p-ATR(T1989), anti-p-DNA-PKcs(S2056), and ␤ -actin.

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Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: Involvement of Host Cellular Factors

Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: Involvement of Host Cellular Factors

A formaldehyde cross-linking ChIP assay was performed to ensure that these identified cellular proteins are indeed asso- ciated with ori-Lyt DNA in the virus context in cells. In brief, protein-DNA cross-linking was induced by addition of formal- dehyde to living TPA-induced BCBL-1 cells. Chromatin from these cells was fragmented by sonication, and the resulting material was immunoprecipitated with specific antibodies against each of the viral and cellular proteins to be tested. The protein-bound DNAs were quantified by PCR using two pairs of primers designed to amplify the KSHV sequences from nucleotides 23147 to 23405 and from nucleotides 24020 to 24155 within the ori-Lyt (L). The former region includes the K8 binding sites, and the latter region is near the RRE. Findings revealed that (i) both ori-Lyt sequences could be coprecipi- tated by antibodies against three viral proteins, namely, K8, RTA, and ORF57, which are known to be involved in lytic viral DNA replication, but not by preimmune sera (either mouse or rabbit) or antibodies against ORF45 and ORF64 (Fig. 2); (ii) all the cellular proteins tested were found to be associated with at least one of the regions in the ori-Lyt or both; and (iii) RecQL showed the strongest signals for binding to both se- quences of the ori-Lyt among all the cellular proteins tested in the study, suggesting a proximate distance between the protein and ori-Lyt DNA in a replication complex (Fig. 2). Another

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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

A genome wide analysis was performed on the basis of A+T rich islands by the help of bioinformatics tools to find out the AT rich islands (AT content >72% in a 0.5–1.0-kb window). AT-rich islands were shown to reside in intergenic regions and to be over-represented between divergent transcriptional units. The study revealed the presence of 384 such A + T rich regions, which predicted the location of most of the origins within the genome. Average origin density was estimated to be one every 33-kb, with a higher density in centromeric, subtelomeric and mating-type loci (about four-fold higher than the genomic average). Analysis of 20 randomly chosen A+T rich islands by 2D gel technique revealed that ~90% of them (18 islands) function as efficient replication origins in their native locations suggesting that, out of 384 A+T islands, 345 may function as origins in the genome (Segurado et al., 2003). These A + T rich sequences are probably important for the binding of S. pombe ORC (SpORC). The SpORC4 protein specifically binds to AT hook domain (Lee et al., 2001, Kong and DePamphilis 2001). It was shown that the properties of known fission yeast origins were similar to fission yeast intergenic regions in general (Dai et al., 2005). Out of 26 intergenic regions tested on plasmids for origin function, four functioned as monomers and 10 functioned as dimers, leading to the suggestion that approximately half of fission yeast 4978 intergenic regions may have potential origin function. A lack of a consensus sequence was also noted, while AT content and intergenic sizes were suggested to constitute the major determinants of origin activity. Based on these findings, a hypothesis was formulated, suggesting that DNA replication is stochastic in nature and out of many potential origins across the fission yeast genome, only a few will fire in any given cell cycle (Dai et al., 2005).

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Analysis of an Autographa californica Nucleopolyhedrovirus lef-11 Knockout: LEF-11 Is Essential for Viral DNA Replication

Analysis of an Autographa californica Nucleopolyhedrovirus lef-11 Knockout: LEF-11 Is Essential for Viral DNA Replication

To generate a recombinant AcMNPV BACmid containing a lef-11 knockout, we used a modification of a method described by Bideshi and Federici (4) for recombination in E. coli. The AcMNPV BACmid genome used in these studies was originally described as bMON14272 by Luckow and coworkers (21) and is commercially available (Invitrogen Life Technologies). Transfer vector pGEM72 (f⫹)polyAie1Ppp31GUSorf38CAT was digested with ApaI-NsiI. The resulting purified linear 6.558-kbp fragment containing the polyA-GUS-CAT-ie1 pro- moter cassette plus flanking regions from the lef-11 locus was cotransformed with the bMON14272 BACmid DNA into E. coli strain BJ5183 (Stratagene, Inc.). After overnight incubation in SOC, cells were plated onto Luria-Bertani (LB) agar containing 50 ␮g of kanamycin and 30 ␮g of chloramphenicol per ml. Plates were incubated at 37°C for a minimum of 24 h. Colonies that were resistant to kanamycin and chloramphenicol were selected. The presence of the polyA-GUS- CAT-ie1 promoter cassette and the absence of the lef-11 ORF were confirmed by PCR analysis.

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FANCD2 Binds Human Papillomavirus Genomes and Associates with a Distinct Set of DNA Repair Proteins to Regulate Viral Replication

FANCD2 Binds Human Papillomavirus Genomes and Associates with a Distinct Set of DNA Repair Proteins to Regulate Viral Replication

FANCD2 and FANCI protein levels. GAPDH was used as a loading control. (C) Immunofluorescence analysis of control and FANCD2 knockdown cells that were differentiated for 72 h in 1.5 mM calcium medium. Cells were stained with anti-FANCD2 (green) and counterstained with DAPI (blue). (D) CIN612 cells were differentiated for 48 h in 1.5% methylcellulose, and total DNA was isolated from control and shFANCD2 cells. Viral replication was assessed by Southern blot analysis. Similar results were seen using high calcium concentrations to induce differentiation (Fig. S2). Quantification of episomal band intensity was determined by densitometry using Image Lab software and normalized to the undiffer- entiated shGFP-infected sample across three independent experiments. Differences in episomal levels between mock- and shGFP-infected cells were not statistically significant. Error bars represent the standard deviations between experiments. A standard Student’s t test was used to determine statistical significance. *, P ⱕ 0.05. (E) The ratio of episomal DNA in undifferentiated and differentiated samples was calculated to determine fold amplification in knockdown and control cells. Error bars represent the standard deviations between experiments. ns, not significant. (F) Control and shFANCD2 cells were differentiated for 48 h in 1.5% methylcellulose. Total RNA was isolated, and early transcript expression was determined by Northern blot analysis. The Northern blot shows expression of the primary early transcript E6*E7 E1^E4 E5. (G) CIN612 cells that stably express either control or shFANCD2 were seeded at 5 ⫻ 10 4 into each well of a 6-well cell culture dish. Cells

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Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

To form the MRN complex, Nbs1 binds Mre11, which binds to Rad50 (40, 62). Since we were able to immunoprecipitate Nbs1 with E7, we next wanted to determine if E7 interacts with other components of the MRN complex. For this, we immunoprecipi- tated HA-tagged HPV31 E7 or TAP-tagged HPV16 E7 from ly- sates harvested from U20S cells and performed Western blot anal- ysis for Mre11 and Rad50. Interestingly, as shown in Fig. 4A, we found that both HPV31 E7 and HPV16 E7 could immunoprecipi- tate with Rad50, but neither interacted with Mre11. However, we found that the presence of E7 did not disrupt formation of the MRN complex, as Nbs1 was still able to interact with Rad50 and Mre11 (Fig. 4B). Rather, our results indicate that the presence of E7 increases the formation of the MRN complex, which may result from increased Mre11 expression. We also found that the forma- tion of the MRN complex is not disrupted in HPV-infected cells (see Fig. 8B) or cells stably expressing HPV31 E7 (data not shown). Nbs1 is necessary for productive viral replication. Previously, we demonstrated that inhibition of ATM kinase activity has min- imal effect on the ability of HPV to be maintained as an episome (12). However, in addition to being essential for ATM activation in response to DSBs, Nbs1 can also mediate ATR activation (63, 64), which could potentially be important for episomal mainte- nance. To examine the effect of Nbs1 depletion on episomal main- tenance, we transduced HPV31-positive CIN612 9E cells with a Scramble control shRNA or a previously validated Nbs1 shRNA (65) and generated stable cell lines. The cells were routinely pas- saged, and DNA and protein were harvested at every passage. Southern blot analysis was performed to examine the status of the episomal viral DNA. As shown in Fig. 5 (see also Fig. 12A), in two independently derived CIN612 9E lines, there was no significant

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Adenovirus E1A Oncogene Induces Rereplication of Cellular DNA and Alters DNA Replication Dynamics

Adenovirus E1A Oncogene Induces Rereplication of Cellular DNA and Alters DNA Replication Dynamics

Our data suggest that the grossly altered DNA replication pat- terns, including DNA rereplication in E1A-positive cells, result in replication stress, as evidenced by the activation of a DNA damage response (DDR). In eukaryotic cells, when cells encounter DNA damage, two parallel check point signaling pathways are activated, both of which inhibit Cdc25, a positive modulator of cell cycle progression. The first signaling module, ATM/Chk2, is activated after DNA double-strand breaks, whereas the ATR/Chk1 module FIG 5 Summary of the DNA combing analysis, showing changes in fork velocity, replicon length, and interorigin distance of DNA replication patterns in E1A-expressing cells compared to serum-stimulated cells at both early (A) and late (B) time points. (A) Combing analysis of DNA at early S phase. Top: analysis of the fork velocity (Fv) distribution of the two samples calculated as (IdU Fv ⫹ CldU Fv)/2. Middle: analysis of the total replicon length. Bottom: analysis of the interorigin distance. For each sample, the mean value (Mean) and the number of observed events (N) are reported. The error bars indicate the 95% confidence intervals. Data sets were analyzed with the nonparametric test for unpaired data (Mann-Whitney). E1A-expressing cells and b-gal samples showed P ⬍ 0.001 for fork velocity and replicon length and P ⬍ 0.01 for interorigin distance. (B) Summary of DNA combing assay results determined for DNA samples isolated from cells harvested in late S phase. Details of the experiment were as described above with the exception that infection was allowed to proceed for 48 h. Top: analysis of the fork velocity distribution. Middle: analysis of the total replicon length. Bottom: analysis of the interorigin distance. Analysis of data sets and P values was performed as described for panel A.

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Chromosomal Lesion Suppression and Removal in Escherichia coli via Linear DNA Degradation

Chromosomal Lesion Suppression and Removal in Escherichia coli via Linear DNA Degradation

Figure 3.—pRecBCD⫹ plasmid fully complements the linear DNA degradation defect and partially complements the recombi- national repair defect of recBCD mutant cells. The assays in A–C employ wild type, ⌬recBCD mutant cells, and ⌬recBCD mutant complemented with the vector alone or with the pRecBCD⫹ plasmid. The viability assay in D employs wild-type, ⌬recA, ⌬recBCD, and ⌬recA ⌬recBCD strains. (A) Linear DNA degradation capacity, as gauged by plating efficiency of T4 2 mutant phage. (B) Recombinational repair capacity, as gauged by survival of UV irradiation. (C) Recombination repair capacity vs. linear DNA degradation activity in functional interaction with each other, as gauged by a two-marker P1 transduction. (D) Viability expressed as colony-forming efficiency. The strains are: AB1157, the wild-type control; ⌬recA, JC10287; ⌬recBCD, JB1; ⌬recA ⌬recBCD, AM1. The plasmids are: vector, pWSK29; pRecBCD⫹, pAMP1.

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Current versus future reproduction and longevity: a re evaluation of predictions and mechanisms

Current versus future reproduction and longevity: a re evaluation of predictions and mechanisms

To determine whether a reproductive event has had a negative, neutral or positive impact on bioenergetic capacity, it is necessary to measure mitochondrial respiratory function to determine how relative ROS levels, oxidative damage, antioxidant production, repair, replication errors and mitochondrial biogenesis have affected bioenergetic capacity. Several techniques have been used for measuring the functional capacity of mitochondria. Two of most commonly applied methods include the respiratory control ratio (RCR) and the ADP/oxygen ratio (also known as P/O ratio). The RCR is the ratio of state 3 (maximum respiratory rate) to state 4 (basal respiratory rate) respiration, representing the ability of the mitochondria to increase respiratory rate in response to newly available adenosine diphosphate (ADP; Brand and Nicholls, 2011). This method is valuable because it is sensitive to any changes in redox state and the functional capacity of the ETS. However, as substrate must be added to the mitochondria to fuel respiration, the responses that are measured will be substrate specific, and may or may not directly reflect the nutrients used to fuel activity in vivo (Kuzmiak et al., 2012). Moreover, because RCR is a ratio between state 3 and state 4 respiration, simultaneous changes to both state 3 and state 4 could be overlooked by RCR. As a result, we encourage researchers to report both state 3 and state 4 levels with RCR. Another method often used to investigate mitochondrial function is the P/O ratio. The P/O ratio measures the relative coupling efficiency of the mitochondria, which constrains mechanistic models of the electron-transport chain and ATP synthase (Brand, 2005; Salin et al., 2015a). Mitochondrial coupling may contribute to individual differences in performance for select life-history variables, such as growth and ageing (Speakman et al., 2004; Salin et al., 2012; but see Stier et al., 2014) – its role in the interaction among life-history variables warrants

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