Guanine quadruplex structures localize to heterochromatin

Abstract

Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochromatin. Polytene chromosomes from Drosophila salivary glands showed bands that co-localized with heterochromatin proteins HP1 and the SNF2 domain-containing protein SUUR. Staining was retained in SUUR knock-out mutants but lost upon overexpression of SUUR. Somatic cells in Macrostomum lignano were strongly labeled, but pluripotent stem cells labeled weakly. Similarly, germline stem cells in Drosophila ovaries were weakly labeled compared to most other cells. The unexpected presence of G4 structures in heterochromatin and the difference in G4 staining between somatic cells and stem cells with germline DNA in ciliates, flatworms, flies and mammals point to a conserved role for G4 structures in nuclear organization and cellular differentiation.

Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells

“…In the virions the viral genome is linear and double-stranded and no replication events occur. The presence of G4s at this level is compatible with the reported induction of G4s under molecular crowded conditions (53) and with the recently provided evidence that G4s are also present in post-mitotic DNA (21). …”

“…Attempts to characterize the DNA enriched for 1H6 binding sites have thus far been unsuccessful (8,21), however, since (i) the HSV-1 genome is extremely rich in G-rich regions that fold in G4 in vitro (17); (ii) a G4 ligand was able to suppress viral DNA replication and infectivity (17); (iii) ACV and PAA, compounds that selectively inhibit the viral DNA polymerase, decrease the amount of G4s in the infected cell (shown here); (iv) G4s in infected cells show striking virus cycle- and virus amount-dependence (shown here); (v) G4s in infected cells associate with viral RCs and are present in immature virions before and after budding at the nuclear membrane (shown here) convincingly indicate that the observed G4s are viral structures.…”

“…The mouse monoclonal 1H6 antibody has been previously reported (8,21). The rabbit 3–83 antiserum specific for ICP8 was a kind gift from David M. Knipe (Harvard Medical School, Boston, USA) (22) anti-HSV-1 ICP8 monoclonal antibody conjugated with FITC was obtained from Santa Cruz Biotechnology Inc.…”

“…Immuno-EM samples were prepared as previously described (21). At 8 and 15 h.p.i., cells infected with wt HSV-1 at MOI 5 were washed with PBS 1× and fixed in 2.5% glutaraldehyde buffered in 0.1 M cacodylate buffer (Sigma Aldrich) for 1 h at 4°C.…”

The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment

“…It has since been demonstrated that ChlR1 is important for heterochromatin organization and that its depletion causes decreased chromatin compaction at pericentric and telomeric chromosomal regions (30). Furthermore, in vitro assays showed that the ChlR1 helicase efficiently unwinds DNA structures that contain G-quadruplex (G4) structures (31), which are frequently found in heterochromatin (32). This ability to resolve G4 structures that inevitably stall replication forks may help to explain why ChlR1-deficient cells are highly sensitive to DNA cross-linking agents, such as cisplatin and mitomycin C, that stall the replication machinery (33).…”