Cooperation of DNA-PKcs and WRN helicase in the maintenance of telomeric D-loops

Werner syndrome is an inherited human progeriod syndrome caused by mutations in the gene encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA helicase and exonuclease activities, and is suggested to have roles in many aspects of DNA metabolism, including DNA repair and telomere maintenance. The DNA-PK complex also functions in both DNA double strand break repair and telomere maintenance. Interaction between WRN and the DNA-PK complex has been reported in DNA double strand break repair, but their possible cooperation at telomeres has not been reported. This study analyzes thein vitro and in vivo interaction at the telomere between WRN and DNA-PKcs, the catalytic subunit of DNA-PK. The results show that DNA-PKcs selectively stimulates WRN helicase but not WRN exonuclease in vitro, affecting that WRN helicase unwinds and promotes the release of the full-length invading strand of a telomere D-loop model substrate. In addition, the length of telomeric G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is reversed by overexpression of WRN helicase. These results suggest that WRN and DNA-PKcs may cooperatively prevent G-tail shortening in vivo

Serines 440 and 467 in the Werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks

WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and −467 in WRN as major phosphorylation sites mediated by DNA-PK. In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and −467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair

papillomavirus types 16 and 18 mediated by

Replication interference between huma

Genomic Instability and Cancer Risk Associated with Erroneous DNA Repair

Many cancers develop as a consequence of genomic instability, which induces genomic rearrangements and nucleotide mutations. Failure to correct DNA damage in DNA repair defective cells, such as in BRCA1 and BRCA2 mutated backgrounds, is directly associated with increased cancer risk. Genomic rearrangement is generally a consequence of erroneous repair of DNA double-strand breaks (DSBs), though paradoxically, many cancers develop in the absence of DNA repair defects. DNA repair systems are essential for cell survival, and in cancers deficient in one repair pathway, other pathways can become upregulated. In this review, we examine the current literature on genomic alterations in cancer cells and the association between these alterations and DNA repair pathway inactivation and upregulation

USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair

Identification of TRAPPC8 as a Host Factor Required for Human Papillomavirus Cell Entry

<div><p>Human papillomavirus (HPV) is a non-enveloped virus composed of a circular DNA genome and two capsid proteins, L1 and L2. Multiple interactions between its capsid proteins and host cellular proteins are required for infectious HPV entry, including cell attachment and internalization, intracellular trafficking and viral genome transfer into the nucleus. Using two variants of HPV type 51, the Ma and Nu strains, we have previously reported that MaL2 is required for efficient pseudovirus (PsV) transduction. However, the cellular factors that confer this L2 dependency have not yet been identified. Here we report that the transport protein particle complex subunit 8 (TRAPPC8) specifically interacts with MaL2. TRAPPC8 knockdown in HeLa cells yielded reduced levels of reporter gene expression when inoculated with HPV51Ma, HPV16, and HPV31 PsVs. TRAPPC8 knockdown in HaCaT cells also showed reduced susceptibility to infection with authentic HPV31 virions, indicating that TRAPPC8 plays a crucial role in native HPV infection. Immunofluorescence microscopy revealed that the central region of TRAPPC8 was exposed on the cell surface and colocalized with inoculated PsVs. The entry of Ma, Nu, and L2-lacking PsVs into cells was equally impaired in TRAPPC8 knockdown HeLa cells, suggesting that TRAPPC8-dependent endocytosis plays an important role in HPV entry that is independent of L2 interaction. Finally, expression of GFP-fused L2 that can also interact with TRAPPC8 induced dispersal of the Golgi stack structure in HeLa cells, a phenotype also observed by TRAPPC8 knockdown. These results suggest that during viral intracellular trafficking, binding of L2 to TRAPPC8 inhibits its function resulting in Golgi destabilization, a process that may assist HPV genome escape from the trans-Golgi network.</p></div

TRAPPC8 coprecipitation with L2.

<p>(A) Schematic diagram of HPV51 L2s. The percentage of cells expressing GFP after inoculation with PsVs containing GFP-expression plasmid, HPV51 Ma L1, and each L2 are shown on the right. (B) Electrophoresis of subcellular proteins that coprecipitated with FLAG-tagged 51NuL2 (51NuL2-FLAG) or FLAG-tagged 51MaL2 (51MaL2-FLAG). Proteins were stained with SYPRO Ruby. (C) TRAPPC8 and TRAPPC12 coprecipitated with FLAG-tagged HPV51 L2s (51NuL2-FLAG, 51MaL2-FLAG, Ch5L2-FLAG, Ch4L2-FLAG), HPV16 L2 (16L2-FLAG), and HPV31 L2 (31L2-FLAG). Precipitated L2s were stained with SYPRO Ruby (left panel). Subcellular proteins coprecipitated with L2s were analyzed by Western blotting using anti-TRAPPC8 antibody (Anti-P880/894) (middle panel) or anti-TRAPPC12 antibody (Anti-TTC15) (right panel). Asterisks: unknown proteins cross reactive with the antibodies.</p

Effects of TRAPPC8 knockdown on PsV internalization.

<p>(A) HeLa cells transfected with control or TRAPPC8 siRNA (KIAA1012-04) were inoculated with PsV (MOI of ∼2000 particles/cell) and incubated for 1 h at 4°C. After washing with PBS, the cells were incubated in medium at 37°C for 0, 1, 2, 4, or 8 h. The cells were detached with PBS containing EDTA (trypsin –) or PBS containing trypsin and EDTA (trypsin +). The detached cells were lysed and boiled. HPV51 L1, TRAPPC8, and α-tubulin were detected by Western blotting using anti-51L1 antiserum, anti-TRAPPC8 (anti-N1/603), and anti-α-tubulin antibodies, respectively. HPV16 and HPV31 L1s were detected using anti-HPV16L1 antibody. Asterisks: unknown protein that reacted with the anti-HPV16L1 antibody. (B) Quantification of trypsin-resistant, full-length L1 in cells. HeLa cells transfected with control (black circle) or TRAPPC8 siRNA (KIAA1012-04) (white circle) were inoculated with 51PsVMaL2, 51PsVNuL2, 51PsVL2-, 16PsV, 16PsVL2–, 31PsV, or 31PsVL2– (MOI of ∼2000 particles/cell). After washing with PBS, the cells were incubated in the medium at 37°C for 0, 1, 2, 4, or 8 h. The cells were detached with PBS containing trypsin, and L1 in the cell lysates was detected by Western blotting (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080297#pone.0080297.s003" target="_blank">Figure S3B</a>). Trypsin-resistant, full-length L1 was quantified using a Typhoon 9410 imager. The data for each PsV were obtained from one experiment.</p

Effects of TRAPPC8 knockdown on gene transduction of PsVs and infection with authentic HPV31 virions.

<p>(A) HeLa cells were transfected with individual TRAPPC8 siRNAs (KIAA1012-01, -02, -03, or -04). At 2 days after siRNA transfection the cells were inoculated with 51PsVMaL2 (MOI of ∼50 particles/cell). Following incubation for 2 days, the number of cells expressing GFP was measured by flow cytometry (lower left panel). Top panels: Western blotting of cells transfected with the indicated TRAPPC8 siRNAs using anti-TRAPPC8 antibody (Anti-N1/603) and anti-α-tubulin antibodies as a loading control. Lower right panel: viability of HeLa cells transfected with the indicated TRAPPC8 siRNAs was analyzed using a WST-1 cell proliferation assay at 96 h post-transfection. (B) HeLa cells were transfected with the indicated TRAPPC8 siRNAs (KIAA1012-03, or -04). At two days after siRNA transfection the cells were inoculated with 51PsVMaL2, 16PsV or 31PsV (MOI of ∼50 particles/cell). Following incubation for 2 days, the number of cells expressing GFP was measured by flow cytometry. (C) HaCaT cells were transfected with the indicated TRAPPC8 siRNAs (KIAA1012-03 or -04). At two days after siRNA transfection the cells were inoculated with HPV31b virions prepared from CIN612-9E raft tissues. Following incubation for 3 days, E1ˆE4 viral transcript was quantified by RT-qPCR. The level of the HPV31E1ˆE4 transcript was normalized to that of β-actin mRNA. E1ˆE4 transcript relative to beta-actin mRNA in control siRNA transfected cells was set as 100%. Upper panels: Western blot of HaCaT cells at 48 h post-transfection with the indicated TRAPPC8 siRNAs using anti-N1/603 and anti-α-tubulin antibody. (D) Effects of TRAPPC12 knockdown on gene transduction with 51PsVMaL2. HeLa cells were transfected with control, individual TRAPPC12 siRNAs (TTC15-01, -02, -03, or -04) or TRAPPC8 siRNA (KIAA1012-04). At 2 days after siRNA transfection the cells were inoculated with 51PsVMaL2 (MOI of ∼50 particles/cell). Following incubation for 2 days, the number of cells expressing GFP was measured by flow cytometry (left lower panel). Lysates prepared from HeLa cells at 48 h post-transfection were analyzed by Western blot using anti-TRAPPC12 antibody (left upper panel) or anti-TRAPPC8 antibody (anti-N1/603) (right panel). Alpha-tubulin was detected as a loading control. All experiments (A to D) were performed in triplicate. Error bars indicate standard deviations.</p