Biomedical subjects

Konstantin Brodolin

Publications and source records attributed to Konstantin Brodolin.

4 recordsLinked to original sources

PubMed2006

HoxB domain induction silences DNA replication origins in the locus and specifies a single origin at its boundary.

In multicellular organisms, changes in the DNA replication programme could act to integrate differentiation with cell division in various developmental and transcriptional contexts. Here, we have addressed the use of DNA replication origins during differentiation in the HoxB domain-a cluster of nine genes developmentally regulated in a collinear manner. In undifferentiated mouse P19 cells, we detected several DNA replication origins in the 100 kb HoxB locus, indicating a relaxed origin use when the locus is transcriptionally silent. By contrast, in retinoic-acid-induced differentiated cells, when HoxB transcription is activated, a general silencing of DNA replication origins occurs in the locus except one located downstream of Hoxb1, at the 3' boundary of the HoxB domain. Silencing of the replication origins is associated with histone hyperacetylation, whereas the active Hoxb1 origin persists as a hypoacetylated island. These findings provide direct evidence for the differentiated use of origins in HoxB genes, and we suggest that this regulation might contribute to the regulated expression of HoxB genes during development.

Acetylation↗

PubMed2005

Remodeling of the sigma70 subunit non-template DNA strand contacts during the final step of transcription initiation.

Transcription initiation in bacteria requires melting of approximately 13 bp of promoter DNA. The mechanism of the melting process is not fully understood. Escherichia coli RNA polymerase bearing a deletion of the beta subunit lobe I (amino acid residues 186-433) initiates melting of the -10 promoter element but cannot propagate the melting downstream, towards the transcription initiation start site (+1). However, in the presence of nucleotides, stable downstream melting is induced. Here, we studied lacUV5 promoter complexes formed by the mutant enzyme by cross-linking RNA polymerase subunits to single-stranded DNA in the transcription bubble. In the absence of NTPs, a contact between the sigma70 subunit and the non-template strand of the -10 promoter element was detected. This contact disappeared in the presence of NTPs. Instead, a new sigma70-DNA contact as well as stable beta' and beta subunit contacts with the non-template DNA downstream of the -10 promoter element were established. In terms of the two-step (upstream initiation/downstream propagation) model of promoter melting, our data suggest that beta lobe I induces the propagation of promoter melting by directing downstream promoter DNA duplex towards the downstream DNA-binding channel (beta' clamp). Establishment of downstream contacts leads to remodeling of upstream interactions between sigma70 and the -10 promoter element that might facilitate promoter escape and sigma release.

DNA, Bacterial↗

PubMed2004

Specification of a DNA replication origin by a transcription complex.

In early Xenopus development, transcription is repressed and DNA replication initiates at non-specific sites. Here, we show that a site-specific DNA replication origin can be induced in this context by the assembly of a transcription domain. Deletion of the promoter element abolishes site-specific initiation, and its relocalization to an ectopic site induces a new origin of replication. This process does not require active transcription, and specification of the origin occurs mainly through a decrease in non-specific initiation at sites distant from the promoter. Finally, chromatin immunoprecipitation experiments suggest that site-specific acetylation of histones favours the selection of the active DNA replication origin. We propose that the specification of active DNA replication origins occurs by secondary epigenetic events and that the programming of chromatin for transcription during development contributes to this selection in higher eukaryotes.

Acetylation↗

PubMed2004

The sigma 70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription.

The sigma(70) subunit of Escherichia coli RNA polymerase (RNAP) is a transcription initiation factor that can also be associated with RNAP during elongation. We provide biochemical evidence that sigma(70) induces a transcription pause at the lacUV5 promoter after RNAP has synthesized a 17-nucleotide transcript. The sigma(70)-dependent pausing requires an interaction between sigma(70) and a part of the lac repressor operator sequence resembling a promoter -10 consensus. The polysaccharide heparin triggers the release of sigma(70) from the paused complexes, supporting the view that during the transition from initiation to elongation the interactions between sigma(70) and core RNAP are weakened. We propose that the binding and retention of sigma(70) in elongation complexes are stabilized by its ability to form contacts with DNA of the transcription bubble. In addition, we suggest that the sigma(70) subunit in the elongation complex may provide a target for regulation of gene expression.

Base Sequence↗