IU Indianapolis ScholarWorks :: Browsing by Subject "DNA replication"

Browsing by Subject "DNA replication"

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Acetylation of E2 by P300 Mediates Topoisomerase Entry at the Papillomavirus Replicon
(American Society for Microbiology, 2019-03-21) Thomas, Yanique; Androphy, Elliot J.; Microbiology and Immunology, School of Medicine
Human papillomavirus (HPV) E2 proteins are integral for the transcription of viral genes and the replication and maintenance of viral genomes in host cells. E2 recruits the viral DNA helicase E1 to the origin. A lysine (K111), highly conserved among almost all papillomavirus (PV) E2 proteins, is a target for P300 (EP300) acetylation and is critical for viral DNA replication (E. J. Quinlan, S. P. Culleton, S. Y. Wu, C. M. Chiang, et al., J Virol 87:1497-1507, 2013, https://doi.org/10.1128/JVI.02771-12; Y. Thomas and E. J. Androphy, J Virol 92:e01912-17, 2018, https://doi.org/10.1128/JVI.01912-17). Since the viral genome exists as a covalently closed circle of double-stranded DNA, topoisomerase 1 (Topo1) is thought to be required for progression of the replication forks. Due to the specific effect of K111 mutations on DNA unwinding (Y. Thomas and E. J. Androphy, J Virol 92:e01912-17, 2018, https://doi.org/10.1128/JVI.01912-17), we demonstrate that the E2 protein targets Topo1 to the viral origin, and this depends on acetylation of K111. The effect was corroborated by functional replication assays, in which higher levels of P300, but not its homolog CBP, caused enhanced replication with wild-type E2 but not the acetylation-defective K111 arginine mutant. These data reveal a novel role for lysine acetylation during viral DNA replication by regulating topoisomerase recruitment to the replication origin.IMPORTANCE Human papillomaviruses affect an estimated 75% of the sexually active adult population in the United States, with 5.5 million new cases emerging every year. More than 200 HPV genotypes have been identified; a subset of them are linked to the development of cancers from these epithelial infections. Specific antiviral medical treatments for infected individuals are not available. This project examines the mechanisms that control viral genome replication and may allow the development of novel therapeutics.
Dynamic regulation of Pif1 acetylation is crucial to the maintenance of genome stability
(Springer, 2021) Ononye, Onyekachi E.; Sausen, Christopher W.; Bochman, Matthew L.; Balakrishnan, Lata; Biology, School of Science
PIF1 family helicases are evolutionarily conserved among prokaryotes and eukaryotes. These enzymes function to support genome integrity by participating in multiple DNA transactions that can be broadly grouped into DNA replication, DNA repair, and telomere maintenance roles. However, the levels of PIF1 activity in cells must be carefully controlled, as Pif1 over-expression in Saccharomyces cerevisiae is toxic, and knockdown or over-expression of human PIF1 (hPIF1) supports cancer cell growth. This suggests that PIF1 family helicases must be subject to tight regulation in vivo to direct their activities to where and when they are needed, as well as to maintain those activities at proper homeostatic levels. Previous work shows that C-terminal phosphorylation of S. cerevisiae Pif1 regulates its telomere maintenance activity, and we recently identified that Pif1 is also regulated by lysine acetylation. The over-expression toxicity of Pif1 was exacerbated in cells lacking the Rpd3 lysine deacetylase, but mutation of the NuA4 lysine acetyltransferase subunit Esa1 ameliorated this toxicity. Using recombinant proteins, we found that acetylation stimulated the DNA binding affinity, ATPase activity, and DNA unwinding activities of Pif1. All three domains of the helicase were targets of acetylation in vitro, and multiple lines of evidence suggest that acetylation drives a conformational change in the N-terminal domain of Pif1 that impacts this stimulation. It is currently unclear what triggers lysine acetylation of Pif1 and how this modification impacts the many in vivo functions of the helicase, but future work promises to shed light on how this protein is tightly regulated within the cell.
Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks
(American Society for Biochemistry and Molecular Biology, 2017-02-17) Kim, Hyun-Suk; Nickoloff, Jac A.; Wu, Yuehan; Williamson, Elizabeth A.; Sidhu, Gurjit Singh; Reinart, Brian L.; Jaiswal, Aruna S.; Srinivasan, Gayathri; Patel, Bhavita; Kong, Kimi; Burma, Sandeep; Lee, Suk-Hee; Hromas, Robert A.; Department of Biochemistry & Molecular Biology, IU School of Medicine
Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5' end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5'-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5' end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5'-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5' end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.
Genome Copy Number Regulates Inclusion Expansion, Septation, and Infectious Developmental Form Conversion in Chlamydia trachomatis
(American Society for Microbiology, 2021-01-11) Brothwell, Julie A.; Brockett, Mary; Banerjee, Arkaprabha; Stein, Barry D.; Nelson, David E.; Liechti, George W.; Microbiology and Immunology, School of Medicine
DNA replication is essential for the growth and development of Chlamydia trachomatis, however it is unclear how this process contributes to and is controlled by the pathogen's biphasic lifecycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature sensitive dnaE mutant (dnaEts ) that exhibits a ∼100-fold reduction in genome copy number at the non-permissive temperature (40°C), but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in dnaEts at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. dnaEts formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernable peptidoglycan and polymerized MreB, the major cell division organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion reversed when dnaEts was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE also restored dnaEts genome replication and inclusion expansion at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe's replicative and infectious forms.SIGNIFICANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and non-infectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen's infectious cycle has not been characterized. We show that genome replication is dispensable for EB to RB conversion, but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe's biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.
Impact of Age and Insulin-Like Growth Factor-1 on DNA Damage Responses in UV-Irradiated Human Skin
(MDPI, 2017-02-26) Kemp, Michael G.; Spandau, Dan F.; Travers, Jeffrey B.; Dermatology, School of Medicine
The growing incidence of non-melanoma skin cancer (NMSC) necessitates a thorough understanding of its primary risk factors, which include exposure to ultraviolet (UV) wavelengths of sunlight and age. Whereas UV radiation (UVR) has long been known to generate photoproducts in genomic DNA that promote genetic mutations that drive skin carcinogenesis, the mechanism by which age contributes to disease pathogenesis is less understood and has not been sufficiently studied. In this review, we highlight studies that have considered age as a variable in examining DNA damage responses in UV-irradiated skin and then discuss emerging evidence that the reduced production of insulin-like growth factor-1 (IGF-1) by senescent fibroblasts in the dermis of geriatric skin creates an environment that negatively impacts how epidermal keratinocytes respond to UVR-induced DNA damage. In particular, recent data suggest that two principle components of the cellular response to DNA damage, including nucleotide excision repair and DNA damage checkpoint signaling, are both partially defective in keratinocytes with inactive IGF-1 receptors. Overcoming these tumor-promoting conditions in aged skin may therefore provide a way to lower aging-associated skin cancer risk, and thus we will consider how dermal wounding and related clinical interventions may work to rejuvenate the skin, re-activate IGF-1 signaling, and prevent the initiation of NMSC.
Insulin-like Growth Factor 1 Receptor Signaling Is Required for Optimal ATR-CHK1 Kinase Signaling in Ultraviolet B (UVB)-irradiated Human Keratinocytes
(American Society for Biochemistry and Molecular Biology, 2017-01-27) Kemp, Michael G.; Spandau, Dan F.; Simman, Richard; Travers, Jeffrey B.; Biochemistry and Molecular Biology, School of Medicine
UVB wavelengths of light induce the formation of photoproducts in DNA that are potentially mutagenic if not properly removed by the nucleotide excision repair machinery. As an additional mechanism to minimize the risk of mutagenesis, UVB-irradiated cells also activate a checkpoint signaling cascade mediated by the ATM and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) kinases to transiently suppress DNA synthesis and cell cycle progression. Given that keratinocytes in geriatric skin display reduced activation of the insulin-like growth factor 1 receptor (IGF-1R) and alterations in DNA repair rate, apoptosis, and senescence following UVB exposure, here we used cultured human keratinocytes in vitro and skin explants ex vivo to examine how IGF-1R activation status affects ATR-CHK1 kinase signaling and the inhibition of DNA replication following UVB irradiation. We find that disruption of IGF-1R signaling with small-molecule inhibitors or IGF-1 withdrawal partially abrogates both the phosphorylation and activation of CHK1 by ATR and the accompanying inhibition of chromosomal DNA synthesis in UVB-irradiated keratinocytes. A critical protein factor that mediates both ATR-CHK1 signaling and nucleotide excision repair is replication protein A, and we find that its accumulation on UVB-damaged chromatin is partially attenuated in cells with an inactive IGF-1R. These results indicate that mutagenesis and skin carcinogenesis in IGF-1-deficient geriatric skin may be caused by defects in multiple cellular responses to UVB-induced DNA damage, including through a failure to properly suppress DNA synthesis on UVB-damaged DNA templates.
Lysine acetylation regulates the activity of nuclear Pif1
(Elsevier, 2020) Ononye, Onyekachi E.; Sausen, Christopher W.; Balakrishnan, Lata; Bochman, Matthew L.; Biology, School of Science
In Saccharomyces cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase, ATPase, and DNA-binding activities, whereas maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity.
Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks
(American Society for Biochemistry and Molecular Biology, 2017-01-27) Kim, Hyun-Suk; Williamson, Elizabeth A.; Nickoloff, Jac A.; Hromas, Robert A.; Lee, Suk-Hee; Biochemistry and Molecular Biology, School of Medicine
Stalling at DNA replication forks generates stretches of single-stranded (ss) DNA on both strands that are exposed to nucleolytic degradation, potentially compromising genome stability. One enzyme crucial for DNA replication fork repair and restart of stalled forks in human is Metnase (also known as SETMAR), a chimeric fusion protein consisting of a su(var)3-9, enhancer-of-zeste and trithorax (SET) histone methylase and transposase nuclease domain. We previously showed that Metnase possesses a unique fork cleavage activity necessary for its function in replication restart and that its SET domain is essential for recovery from hydroxyurea-induced DNA damage. However, its exact role in replication restart is unclear. In this study, we show that Metnase associates with exonuclease 1 (Exo1), a 5'-exonuclease crucial for 5'-end resection to mediate DNA processing at stalled forks. Metnase DNA cleavage activity was not required for Exo1 5'-exonuclease activity on the lagging strand daughter DNA, but its DNA binding activity mediated loading of Exo1 onto ssDNA overhangs. Metnase-induced enhancement of Exo1-mediated DNA strand resection required the presence of these overhangs but did not require Metnase's DNA cleavage activity. These results suggest that Metnase enhances Exo1-mediated exonuclease activity on the lagging strand DNA by facilitating Exo1 loading onto a single strand gap at the stalled replication fork.
The Molecular Mechanism of Break Induced Replication
(2013-02-14) Ayyar, Sandeep; Malkova, Anna
DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.
OB-Folds and Genome Maintenance: Targeting Protein–DNA Interactions for Cancer Therapy
(MDPI, 2021-07-03) Par, Sui; Vaides, Sofia; VanderVere-Carozza, Pamela S.; Pawelczak, Katherine S.; Stewart, Jason; Turchi, John J.; Medicine, School of Medicine
Genome stability and maintenance pathways along with their requisite proteins are critical for the accurate duplication of genetic material, mutation avoidance, and suppression of human diseases including cancer. Many of these proteins participate in these pathways by binding directly to DNA, and a subset employ oligonucleotide/oligosaccharide binding folds (OB-fold) to facilitate the protein-DNA interactions. OB-fold motifs allow for sequence independent binding to single-stranded DNA (ssDNA) and can serve to position specific proteins at specific DNA structures and then, via protein-protein interaction motifs, assemble the machinery to catalyze the replication, repair, or recombination of DNA. This review provides an overview of the OB-fold structural organization of some of the most relevant OB-fold containing proteins for oncology and drug discovery. We discuss their individual roles in DNA metabolism, progress toward drugging these motifs and their utility as potential cancer therapeutics. While protein-DNA interactions were initially thought to be undruggable, recent reports of success with molecules targeting OB-fold containing proteins suggest otherwise. The potential for the development of agents targeting OB-folds is in its infancy, but if successful, would expand the opportunities to impinge on genome stability and maintenance pathways for more effective cancer treatment.

Browsing by Subject "DNA replication"

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