Consequences of Cisplatin Binding on Nucleosome Structure and Dynamics

The effects of cisplatin binding to DNA were explored at the nucleosome level to incorporate key features of the eukaryotic nuclear environment. An X-ray crystal structure of a site-specifically platinated nucleosome carrying a 1,3-cis-{Pt(NH[subscript 3])[subscript 2]}[superscript 2+]-d(GpTpG) intrastrand cross-link reveals the details of how this adduct dictates the rotational positioning of DNA in the nucleosome. Results from in vitro nucleosome mobility assays indicate that a single platinum adduct interferes with ATP-independent sliding of DNA around the octamer core. Data from in vitro transcription experiments suggest that RNA polymerases can successfully navigate along cisplatin-damaged DNA templates that contain nucleosomes, but stall when the transcription elongation complex physically contacts a platinum cross-link located on the template strand. These results provide information about the effects of cisplatin binding to nuclear DNA and enhance our understanding of the mechanism of transcription inhibition by platinum anticancer compounds.National Cancer Institute (U.S.) (Grant CA034992)David H. Koch Cancer Research FundNational Center for Research Resources (U.S.) (Award RR-15301)United States. Dept. of Energy. Office of Basic Energy Sciences (DE-AC02-06CH11357

Structure of duplex DNA containing the cisplatin 1,2-{Pt(NH3)2}2+-d(GpG) crosslink at 1.77 Å resolution

We report the 1.77-Å resolution X-ray crystal structure of a dodecamer DNA duplex with the sequence 5′-CCTCTGGTCTCC-3′ that has been modified to contain a single engineered 1,2-cis-{Pt(NH3)2}2+-d(GpG) cross-link, the major DNA adduct of cisplatin. These data represent a significant improvement in resolution over the previously published 2.6-Å structure. The ammine ligands in this structure are clearly resolved, leading to improved visualization of the cross-link geometry with respect to both the platinum center and to the nucleobases, which adopt a higher energy conformation. Also better resolved are the deoxyribose sugar puckers, which allow us to re-examine the global structure of platinum-modified DNA. Another new feature of this model is the location of four octahedral [Mg(H2O)6]2+ ions associated with bases in the DNA major groove and the identification of 124 ordered water molecules that participate in hydrogen-bonding interactions with either the nucleic acid or the diammineplatinum(II) moiety.National Cancer Institute (U.S.) (grant CA034992)David H. Koch Institute for Integrative Cancer Research at MIT (Koch Fund Fellowship

Estuarine Artificial Reefs to Enhance Seagrass Planting and Provide Fish Habitat

Small 25-m2 artificial reef sets were deployed 1 m deep in Choctawhatchee Bay, FL, to determine the ability of reefs to aid in the establishment of newly planted Ruppia maritima (widgeon grass) while providing habitat for estuarine fishes. Seagrass survival and coverage were examined for reef configurations and compared with control plots. Visual surveys conducted from June 1996 to May 1997 indicated that the artificial reefs had no effect on the survivorship or growth of the planted R. maritima. The artificial reefs attracted juvenile and young adult fishes and had significantly more species, higher diversity, more individuals, and greater total biomass of fishes per area than did the nonreef controls. The 22 fish species observed at the reefs were typical estuarine residents in the area. Young gray snapper, Lutjanus griseus (a recreationally and commercially important species), was abundant at the reefs. Although the artificial reefs did not increase seagrass planting success, these artificial reefs may increase the number of fishes surviving to adulthood by providing protective habitat

A Peptide Core Motif for Binding to Heterotrimeric G Protein α Subunits

Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to G{alpha}i1. The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of G{alpha}i1 and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with G{alpha} subunits representing all four G protein classes, acting as a core motif for G{alpha} interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (G{alpha}i/0-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to G{alpha}i1-3 in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the G{alpha} subunits and excludes association with G{beta}{gamma}. R6A-G{alpha}i1 complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg2+, suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using G{alpha}i1/G{alpha}s chimeras identify two regions of G{alpha}i1 (residues 1–35 and 57–88) as determinants for strong R6A-Gi{alpha}1 interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states