Biomedical subjects

Christopher D Putnam

Publications and source records attributed to Christopher D Putnam.

10 recordsLinked to original sources

PubMed2006

Analysis of gross-chromosomal rearrangements in Saccharomyces cerevisiae.

Cells utilize numerous DNA metabolic pathways and cell-cycle checkpoints to maintain the integrity of their genome. Failure of these mechanisms can lead to genome instability, abnormal cell proliferation, and cell death. This chapter describes a method for the measurement of the rate of accumulating gross-chromosomal rearrangements (GCRs) in haploid cells of the yeast Saccharomyces cerevisiae. The isolation of cells with GCRs relies on the simultaneous loss of two counterselectable markers, CAN1 and URA3, within a nonessential region on the left arm of chromosome V. Healing of DNA breaks by de novo telomere addition, translocations, large interstitial deletions, and chromosome fusion has been detected using a PCR-based procedure for the mapping and amplification of breakpoint junctions, which is also described in detail here. This GCR analysis provides an effective tool for the assessment of the contribution by multiple cellular mechanisms to the maintenance of genome integrity.

Base Sequence↗

PubMed2006

Chromosome healing by de novo telomere addition in Saccharomyces cerevisiae.

The repair of spontaneous or induced DNA damage by homologous recombination (HR) in Saccharomyces cerevisiae will suppress chromosome rearrangements. Alternative chromosome healing pathways can result in chromosomal instability. One of these pathways is de novo telomere addition where the end of a broken chromosome is stabilized by telomerase-dependent addition of telomeres at non-telomeric sites. De novo telomere addition requires the recruitment of telomerase to chromosomal targets. Subsequently, annealing of the telomerase reverse transcriptase RNA-template (guide RNA) at short regions of homology is followed by extension of the nascent 3'-end of the broken chromosome to copy a short region of the telomerase guide RNA; multiple cycles of this process yield the new telomere. Proteins including Pif1 helicase, the single-stranded DNA-binding protein Cdc13 and the Ku heterocomplex are known to participate in native telomere functions and also regulate the de novo telomere addition reaction. Studies of the sequences added at de novo telomeres have lead to a detailed description of the annealing-extension-dissociation cycles that copy the telomerase guide RNA, which can explain the heterogeneity of telomeric repeats at de novo and native telomeres in S. cerevisiae.

Chromosome Breakage↗

PubMed2005

Protein mimicry of DNA and pathway regulation.

The discoveries of DNA mimicry by proteins inspired by Ugi experiments led by Dale Mosbaugh and his colleagues have sparked dramatic insights for our understanding of DNA and protein interactions. Currently only a small number protein mimics of DNA are known or suspected, including Ugi, HI1450, Ocr, TAF1, MfpA, and Dinl. These proteins are structurally diverse, but together they share common themes we define here. These mimics tend to resemble distorted rather than normal B-DNA, possibly to prevent cross-reactions with other DNA metabolizing proteins that should not be inhibited. Side-chain carboxylates of glutamates and aspartates functionally replace phosphates and thereby generate an overall charge pattern resembling the DNA phosphate backbone. Most protein mimics of DNA have strikingly hydrophobic cores that likely stabilize the protein fold despite substantial charge localization and a relatively small internal volume enforced by the restrictions from DNA size. These common characteristics for protein mimicry of DNA should prove useful for future identifications of DNA mimics, which seem likely to be found in bacteriophages, conjugative plasmids, eukaryotic viruses, and transcription machinery. We also suggest approaches to the design of novel DNA mimics to inhibit specific pathways and could be important for basic science applications and for use as therapeutic agents. Moreover, mimicry in general is of critical importance in that it provides an elegant mechanism by which interfaces can be reused to force sequential rather than simultaneous complex formations such as seen in systems involving polar protein assemblies and DNA repair machinery.

Animals↗

PubMed2005

Mutation in Rpa1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice.

Most cancers have multiple chromosomal rearrangements; the molecular mechanisms that generate them remain largely unknown. Mice carrying a heterozygous missense change in one of the DNA-binding domains of Rpa1 develop lymphoid tumors, and their homozygous littermates succumb to early embryonic lethality. Array comparative genomic hybridization of the tumors identified large-scale chromosomal changes as well as segmental gains and losses. The Rpa1 mutation resulted in defects in DNA double-strand break repair and precipitated chromosomal breaks as well as aneuploidy in primary heterozygous mutant mouse embryonic fibroblasts. The equivalent mutation in yeast is hypomorphic and semidominant and enhanced the formation of gross chromosomal rearrangements in multiple genetic backgrounds. These results indicate that Rpa1 functions in DNA metabolism are essential for the maintenance of chromosomal stability and tumor suppression.

Aneuploidy↗

PubMed2005

Saccharomyces cerevisiae as a model system to define the chromosomal instability phenotype.

Translocations, deletions, and chromosome fusions are frequent events seen in cancers with genome instability. Here we analyzed 358 genome rearrangements generated in Saccharomyces cerevisiae selected by the loss of the nonessential terminal segment of chromosome V. The rearrangements appeared to be generated by both nonhomologous end joining and homologous recombination and targeted all chromosomes. Fifteen percent of the rearrangements occurred independently more than once. High levels of specific classes of rearrangements were isolated from strains with specific mutations: translocations to Ty elements were increased in telomerase-defective mutants, potential dicentric translocations and dicentric isochromosomes were associated with cell cycle checkpoint defects, chromosome fusions were frequent in strains with both telomerase and cell cycle checkpoint defects, and translocations to homolog genes were seen in strains with defects allowing homoeologous recombination. An analysis of human cancer-associated rearrangements revealed parallels to the effects that strain genotypes have on classes of rearrangement in S. cerevisiae.

Chromosomal Instability↗

PubMed2004

Chromosome healing through terminal deletions generated by de novo telomere additions in Saccharomyces cerevisiae.

Broken chromosomes healed by de novo addition of a telomere are a major class of genome rearrangements seen in Saccharomyces cerevisiae and similar to rearrangements seen in human tumors. We have analyzed the sequences of 534 independent de novo telomere additions within a 12-kb region of chromosome V. The distribution of events mirrored that of four-base sequences consisting of the GG, GT, and TG dinucleotides, suggesting that de novo telomere additions occur at short regions of homology to the telomerase guide RNA. These chromosomal sequences restrict potential registrations of the added telomere sequence. The first 11 nucleotides of the addition sequences fell into common families that included 91% of the breakpoints. The observed registrations suggest that the 3' end of the TLC1 guide RNA is involved in annealing but not as a template for synthesis. Some families of added sequences can be accounted for by one cycle of annealing and extension, whereas others require a minimum of two. The same pattern emerges for sequences added onto the most common addition sequence, indicating that de novo telomeres are added and extended by the same process. Together, these data indicate that annealing is central to telomerase registration, which limits telomere heterogeneity and resolves the problem of synthesizing Rap1 binding sites by a nonprocessive telomerase with a low-complexity guide RNA sequence.

Chromosome Aberrations↗

PubMed2003

Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures.

Green fluorescent protein has revolutionized cell labeling and molecular tagging, yet the driving force and mechanism for its spontaneous fluorophore synthesis are not established. Here we discover mutations that substantially slow the rate but not the yield of this posttranslational modification, determine structures of the trapped precyclization intermediate and oxidized postcyclization states, and identify unanticipated features critical to chromophore maturation. The protein architecture contains a dramatic approximately 80 degrees bend in the central helix, which focuses distortions at G67 to promote ring formation from amino acids S65, Y66, and G67. Significantly, these distortions eliminate potential helical hydrogen bonds that would otherwise have to be broken at an energetic cost during peptide cyclization and force the G67 nitrogen and S65 carbonyl oxygen atoms within van der Waals contact in preparation for covalent bond formation. Further, we determine that under aerobic, but not anaerobic, conditions the Gly-Gly-Gly chromophore sequence cyclizes and incorporates an oxygen atom. These results lead directly to a conjugation-trapping mechanism, in which a thermodynamically unfavorable cyclization reaction is coupled to an electronic conjugation trapping step, to drive chromophore maturation. Moreover, we propose primarily electrostatic roles for the R96 and E222 side chains in chromophore formation and suggest that the T62 carbonyl oxygen is the base that initiates the dehydration reaction. Our molecular mechanism provides the basis for understanding and eventually controlling chromophore creation.

Biophysical Phenomena↗

PubMed2002

Maintenance of genome stability in Saccharomyces cerevisiae.

Most human cancer cells show signs of genome instability, ranging from elevated mutation rates to gross chromosomal rearrangements and alterations in chromosome number. Little is known about the molecular mechanisms that generate this instability or how it is suppressed in normal cells. Recent studies of the yeast Saccharomyces cerevisiae have begun to uncover the extensive and redundant pathways that keep the rate of genome rearrangements at very low levels. These studies, which we review here, have implicated more than 50 genes in the suppression of genome instability, including genes that function in S-phase checkpoints, recombination pathways, and telomere maintenance. Human homologs of several of these genes have well-established roles as tumor suppressors, consistent with the hypothesis that the mechanisms preserving genome stability in yeast are the same mechanisms that go awry in cancer.

Base Sequence↗

PubMed2002

Structure and function correlation in histone H2A peptide-mediated gene transfer.

Histone H2A has been found to be efficient in DNA delivery into a number of cell lines. We have reasoned that this DNA-delivery activity is mediated by two mechanisms: (i) electrostatically driven DNA binding and condensation by histone and (ii) nuclear import of these histone H2A.DNA polyplexes via nuclear localization signals in the protein. We have identified a 37-aa N-terminal peptide of histone H2A that is active in in vitro gene transfer. This peptide can function as a nuclear localization signal and can bind DNA. Amino acid substitutions that replace positively charged residues and/or DNA-binding residues of this peptide obliterate transfection activity. The introduction of a proline in the first turn of an alpha-helix of this 37-mer obliterates transfection activity, suggesting that the integrity of the alpha-helical structure of the N-terminal region of histone H2A is related to its transfection activity.

Amino Acid Sequence↗

PubMed2002

DNA double-strand break repair from head to tail.

DNA double-strand break repair is a complex process that requires multiple enzymatic and structural activities to rejoin or repair the broken DNA ends using one of several repair pathways. These enzymatic and structural activities include end detection, end processing and alignment of DNA ends. Recent structural and functional studies of the DNA double-strand break repair factors Mre11/Rad50, Ku70/80 and Xrcc4 show how these enzymes combine and assemble both enzymatic and structural activities in DNA double-strand break repair.

Adenosine Triphosphate↗