Biomedical subjects

I Chesnokov

Publications and source records attributed to I Chesnokov.

6 recordsLinked to original sources

PubMed2001

Functional analysis of mutant and wild-type Drosophila origin recognition complex.

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.

Adenosine Triphosphatases↗

PubMed1999

Assembly of functionally active Drosophila origin recognition complex from recombinant proteins.

In eukaryotes the sites for the initiation of chromosomal DNA replication are believed to be determined in part by the binding of a heteromeric origin recognition complex (ORC) to DNA. We have cloned the genes encoding the subunits of the Drosophila ORC. Each of the genes is unique and can be mapped to discrete chromosomal locations implying that the pattern and developmental regulation of origin usage in Drosophila is not regulated solely by a large family of different ORC proteins. The six-subunit ORC can be reconstituted with recombinant proteins into a complex that restores DNA replication in ORC-depleted Drosophila or Xenopus egg extracts.

Amino Acid Sequence↗

PubMed1997

Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes.

The origin recognition complex (ORC) is required to initiate eukaryotic DNA replication and also engages in transcriptional silencing in S. cerevisiae. We observed a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalized with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 were found in physical complex. The association was shown biochemically to require the chromodomain and shadow domains of HP1. The amino terminus of DmORC1 contained a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Finally, heterozygous DmORC2 recessive lethal mutations resulted in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors.

Amino Acid Sequence↗

PubMed1996

Flanking sequences of an Alu source stimulate transcription in vitro by interacting with sequence-specific transcription factors.

An Alu source gene, called the EPL Alu, was previously isolated by a phylogenetic strategy. Sequences flanking the EPL Alu family member stimulate its RNA polymerase III (Pol III) template activity in vitro. One cis-acting element maps within a 40-nucleotide region immediately upstream to the EPL Alu. This same region contains an Ap1 site which, when mutated, abolishes the transcriptional stimulation provided by this region. The flanking sequence, as assayed by gel mobility shift, forms sequence-specific complexes with several nuclear factors including Ap1. These results demonstrate that an an ancestral Alu source sequence fortuitously acquired positive transcriptional control elements by insertion into the EPL locus, thereby providing biochemical evidence for a model which explains the selective amplification of Alu subfamilies.

Base Sequence↗

PubMed1996

p53 inhibits RNA polymerase III-directed transcription in a promoter-dependent manner.

Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation.

Animals↗

PubMed1991

Binding specificity of human nuclear protein interacting with the Alu-family DNA repeats.

We have demonstrated earlier that human cells contain nuclear protein interacting with conserved GC-rich sequence motifs of human Alu-family DNA repeats. One of these sequences is located in the region between elements A and B of bipartite RNA polymerase III promoter of Alu (AB-region). In this study we have used a DNase I footprinting assay with an Alu restriction subfragment covering AB-region, as well as a gel mobility shift assay with appropriate synthetic oligonucleotides to analyse in more detail the interaction of the protein with AB-region. We have also used antibodies raised against a zinc-finger peptide to examine the presence of a zinc-finger in the Alu-binding protein. The results indicate that AGG triplets may be important for high-affinity binding of the protein to DNA, and that the Alu-binding protein is a zinc-finger protein.

Amino Acid Sequence↗