Novel Mechanisms through which translesion synthesis protects genome integrity

Exposure to ubiquitous environmental carcinogens, such as polycyclic aromatic hydrocarbons and UV light, is a major cause of human disease. It is well accepted that genetic mutations are an important step in the development of cancer. It has become clear that such mutations are introduced in part by error-prone DNA polymerases. In response to many environmental genotoxins, eukaryotic cells have evolved alternative methods of replicating damaged DNA via the Translesion DNA synthesis (TLS) Polymerases, consisting of DNA Polymerase eta (Polη), DNA Polymerase kappa (Polκ), DNA Polymerase iota (Polι), and Rev1. TLS is a DNA damage tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome Xeroderma Pigmentosum Variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates PCNA, but the basis for recruitment of Rad18 to PCNA is poorly understood. This dissertation studies several aspects of regulatory mechanisms that contribute to the damage-induced activation of Rad18 E3 ligase activity at PCNA. First, we report a novel role for Polη, the XPV gene product that is mutated in XPV, in targeting Rad18 to PCNA to initiate TLS. Using structure-function analyses and immunofluorescence microscopy, we identified a C-terminal domain of Polη that physically bridges Rad18 and PCNA to facilitate redistribution of Rad18 to stalled replication forks and promote PCNA monoubiquitination. This scaffold function is unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length, catalytically inactive Polη exhibit increased recruitment of error-prone TLS Polymerases after UV irradiation, indicating that maintaining the bridging function of Polη in the absence of its catalytic activity greatly predisposes to mutagenesis. These findings define a molecular basis for TLS pathway activation and provide a new mechanism for mutagenesis and genomic instability in XPV individuals. Next, this dissertation reports novel mechanisms of regulating TLS via stress-activated protein kinase (SAPK) phosphorylation of Rad18 and via Chk1-dependent phosphorylation events. Finally, this dissertation presents data indicating that TLS is involved in the tolerance of oncogene-induced replication stresses and potentially oncogene-induced mutagenesis.Doctor of Philosoph

A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks

Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polη), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polη binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polη exhibit increased recruitment of other error-prone TLS polymerases (Polκ and Polι) after UV irradiation. These results define a novel non-catalytic role for Polη in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals

Phytosociological analysis of restored and managed grassland habitat within an urban national park

Floyd Bennett Field (FBF), 579 ha in extent, is a division of Gateway National Recreation Area. It is the site of a former airfield, constructed by filling salt marshes with dredged materials. Except for the portion known locally as the “North Forty,” all sections of FBF have been cut over to maintain low vegetation. A grassland management plan (GRAMP) for 165 ha was initiated in 1986, to maintain habitats for open-country birds. Over the next few years, encroaching woody vegetation was removed manually and mechanically from the management area. Since then, it has been maintained as a grassland and receives annual mowing, as well as continued manual removal of the larger woody sprouts. A portion of the GRAMP management area (III) was selected for intensive study of vegetation composition. A grid system was created and vegetation cover was estimated in 127, 1 m × 1 m quadrats. The quadrats were subjected to cluster analysis (CA). Eleven clusters were recognized. These clusters were treated as “plant associations.” The following types were distinguished: (native) little bluestem–dewberry grassland, six-weeks fescue annual grassland, a grass marsh, a rush marsh, a switchgrass dry grassland, and a deer-tongue panicgrass grassland; (exotic) mugwort herbland, oriental bittersweet-Japanese honeysuckle vineland, Kentucky bluegrass-mixed grassland, Japanese knotweed tall herbland, and spotted knapweed-common St. Johnswort herbland. The little bluestem–dewberry association accounted for nearly half of all quadrats; six subclusters were recognized. The plant associations determined by CA were compared with plant lists compiled during traverses of all of the map categories in the six GRAMP Areas (I, II, III, IV, V, VI). A table was created to relate the quantitative data of the plant associations to the appropriate map categories. A nonmetric multidimensional scaling ordination (NMDS) was performed on the quadrat data. Finally, the plant associations were compared with those described in the literature of local vegetation studies. The mowing program has been effective in decreasing woody plant cover and has permitted the invasion of a few taxa into monospecific communities, but attendant disturbance of the substrate is likely to cause an increase in exotic plant taxa. As earlier studies noted, mowing has caused the increase in cover of sod-forming grass, and bare ground has virtually disappeared in the managed area. This has negative implications for the maintenance of those grassland bird species that require open ground for nesting

DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

The mechanisms by which neoplastic cells tolerate oncogene-induced DNA replication stress are poorly understood. Cyclin-dependent kinase 2 (CDK2) is a major mediator of oncogenic DNA replication stress. In this study, we show that CDK2-inducing stimuli (including Cyclin E overexpression, oncogenic RAS, and WEE1 inhibition) activate the DNA repair protein RAD18. CDK2-induced RAD18 activation required initiation of DNA synthesis and was repressed by p53. RAD18 and its effector, DNA polymerase κ (Polκ), sustained ongoing DNA synthesis in cells harboring elevated CDK2 activity. RAD18-deficient cells aberrantly accumulated single-stranded DNA (ssDNA) after CDK2 activation. In RAD18-depleted cells, the G2/M checkpoint was necessary to prevent mitotic entry with persistent ssDNA. Rad18 −/− and Polκ −/− cells were highly sensitive to the WEE1 inhibitor MK-1775 (which simultaneously activates CDK2 and abrogates the G2/M checkpoint). Collectively, our results show that the RAD18–Polκ signaling axis allows tolerance of CDK2-mediated oncogenic stress and may allow neoplastic cells to breach tumorigenic barriers

c-Jun N-terminal kinase-mediated Rad18 phosphorylation facilitates Pol recruitment to stalled replication forks

The association of Rad18 with Polη is crucial for efficient translesion synthesis and DNA damage tolerance. Rad18–Polη interactions and UV tolerance depend on JNK-dependent Rad18 phosphorylation. These results provide a new mechanism by which SAPK signaling promotes genome maintenance.The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1–dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage–independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη−PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H2O2-induced DNA damage. UVC and H2O2 treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G0, G1 and S-phase. Rad18 was important for repressing H2O2-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G1, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H2O2-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H2O2-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G1. In contrast with G1-synchronized cultures, S-phase cells were H2O2-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G1 (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase

Sustained Immunogenicity of 2-dose Human Papillomavirus 16/18 AS04-adjuvanted Vaccine Schedules in Girls Aged 9-14 Years: A Randomized Trial

Background: We previously reported the noninferiority 1 month after the last dose of 2-dose human papillomavirus 16/18 AS04-adjuvanted (AS04-HPV-16/18) vaccine schedules at months 0 and 6 (2D_M0,6) and months 0 and 12 (2D_M0,12) in girls aged 9-14 years compared with a 3-dose schedule at months 0, 1, and 6 (3D_M0,1,6) in women aged 15-25 years. Here, we report the results at study end (month 36 [M36]).Methods: Girls were randomized 1:1 and received 2 vaccine doses either 6 months (2D_M0,6) or 12 months apart (2D_M0,12); women received 3 doses at months 0, 1, and 6 (3D_M0,1,6). Endpoints included noninferiority of HPV-16/18 antibodies for 2D_M0,6 versus 3D_M0,1,6; 2D_M0,12 versus 3D_M0,1,6; and 2D_M0,12 versus 2D_M0,6; and assessment of neutralizing antibodies, T cells, B cells, and safety.Results: At M36, the 2D_M0,6 and 2D_M0,12 schedules remained noninferior to the 3D_M0,1,6 schedule in terms of seroconversion rates and 3D/2D geometric mean titers for anti-HPV-16 and anti-HPV-18. All schedules elicited sustained immune responses up to M36.Conclusions: Both 2-dose schedules in young girls remained noninferior to the 3-dose schedule in women up to study conclusion at M36. The AS04-HPV-16/18 vaccine administered as a 2-dose schedule was immunogenic and well tolerated in young girls

SIVA1 directs the E3 ubiquitin ligase RAD18 for PCNA monoubiquitination

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