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R E Wellinger

Publications and source records attributed to R E Wellinger.

9 recordsLinked to original sources
PubMed2001

Nucleosome positioning at the replication fork.

In order to determine the time required for nucleosomes assembled on the daughter strands of replication forks to assume favoured positions with respect to DNA sequence, psoralen cross-linked replication intermediates purified from preparative two-dimensional agarose gels were analysed by exonuclease digestion or primer extension. Analysis of sites of psoralen intercalation revealed that nucleosomes in the yeast Saccharomyces cerevisiae rDNA intergenic spacer are positioned shortly after passage of the replication machinery. Therefore, both the 'old' randomly segregated nucleosomes as well as the 'new' assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands, suggesting that the positioning of nucleosomes is an initial step in the chromatin maturation process.

Base Sequence↗
PubMed2000

DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair.

DNA damage formation and repair are tightly linked to protein-DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication ( ARS1 ). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct inter-actions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication.

DNA Damage↗
PubMed2000

Architecture of the replication fork stalled at the 3' end of yeast ribosomal genes.

Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within an HpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found. In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously synthesized nascent lagging strand was extended three bases farther than the continuously synthesized leading strand. A model explaining these findings is presented. Our analysis included for the first time the use of T4 endonuclease VII, an enzyme recognizing branched DNA molecules. This enzyme cleaved predominantly in the newly synthesized homologous arms, thereby specifically releasing the leading arm.

Base Sequence↗
PubMed1999

Electron microscopic analysis reveals that replication factor C is sequestered by single-stranded DNA.

Replication factor C (RF-C) is a eukaryotic heteropentameric protein required for DNA replication and repair processes by loading proliferating cell nuclear antigen (PCNA) onto DNA in an ATP-dependent manner. Prior to loading PCNA, RF-C binds to DNA. This binding is thought to be restricted to a specific DNA structure, namely to a primer/template junction. Using the electron microscope we have examined the affinity of human heteropentameric RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA. The electron microscopic data indicate that both human heteropentameric RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA. No preferential affinity for the 3' or 5' transition points from single- to double-stranded DNA was evident.

Animals↗
PubMed1998

Histone acetylation facilitates RNA polymerase II transcription of the Drosophila hsp26 gene in chromatin.

A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for those acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, we directly tested for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross-linking as well as by in vitro transcription. While H1-containing control chromatin severely repressed transcription of our model hsp26 gene, highly acetylated chromatin was significantly less repressive. Acetylation of histones, and particularly of histone H4, affected transcription at the level of initiation. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, we found that heat shock factor, a crucial regulator of heat shock gene transcription, profited most from histone acetylation. These experiments demonstrate that histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin.

Acetylation↗
PubMed1997

Nucleosome structure and positioning modulate nucleotide excision repair in the non-transcribed strand of an active gene.

Nucleotide excision repair (NER) is a major pathway to remove pyrimidine dimers (PDs), a class of DNA lesions generated by ultraviolet light. Since folding of DNA into nucleosomes restricts its accessibility and since transcription and DNA repair require access to DNA, nucleosome structure and positioning as well as the transcriptional state may affect DNA repair. We recently determined the chromatin structure of the yeast URA3 gene at high resolution and found multiple positions of nucleosomes as well as strand- and site-specific variation in DNA accessibility to DNase I (internal protected regions). Here, the same high-resolution primer extension technique was used to investigate NER of PDs in the URA3 gene of a mini-chromosome in vivo. In the non-transcribed strand (NTS), fast repair correlates with PD locations in linker DNA and towards the 5' end of a positioned nucleosome. Slow repair correlates with the internal protected region of the nucleosome. This repair heterogeneity reflects a modulation of NER by positioned nucleosomes in the NTS. NER in the transcribed strand (TS) is fast, less heterogeneous and shows no correlation with chromatin structure. Apparently, transcription-coupled repair overrides chromatin modulation of NER in the TS. Heterogeneity in NER generated by chromatin structure on the NTS may contribute to heterogeneity in mutagenesis.

Aldose-Ketose Isomerases↗