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[DNA fragment DARC146 from a complex form of DNA polymerase alpha contains several nuclear protein binding segments]
Abstract:
Earlier, a number of DNA fragments were identified in the complex form of DNA polymerase alpha. One of them, DARC146, can support autonomous replication in mammalian cells. We have subcloned 146 bp from DARC146 (here called DARC146). This fragment has an ability to replicate autonomously in mammalian cells. This ability permits one to speak about DARC146 as a putative replication origin. From this conclusion, we suggest that all signals for initiation of DNA synthesis are located on the nucleotide sequence under study. Here, we have shown that the nuclear extract contains four polypeptides binding specifically to synthetic oligonucleotides covering the AT-rich region of the DARC146 sequence. The first protein is Oct-1, a nuclear transcription-replication factor. The second protein (named p65) binds to the TCTCTTA site of the DARC146 nucleotide sequence. There are two sites for Oct-1 protein and two sites for p65 in the DARC146 fragment. Octamer motifs and sites for p65 are located tandemly side by side. Moreover, we identified 28kDa polypeptide from nuclear matrix which bound to DARC146. Based upon the data presented, we suggest a hypothetical model of the pre-initiation state of the DARC146 sequence.
Insights
A DNA fragment, DARC146, supports autonomous replication in mammalian cells, suggesting it
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA polymerase alpha complex contains DNA fragments with replication capabilities.
- The DARC146 fragment was identified as a potential autonomously replicating sequence in mammalian cells.
Purpose of the Study:
- To investigate the DARC146 fragment as a putative replication origin.
- To identify nuclear proteins that bind to the AT-rich region of DARC146.
- To propose a model for the pre-initiation state of DARC146 replication.
Main Methods:
- Subcloning of the 146 bp DARC146 fragment.
- Analysis of protein binding to synthetic oligonucleotides of the DARC146 AT-rich region using nuclear extracts.
- Identification of nuclear matrix-bound polypeptides.
Main Results:
- The DARC146 fragment demonstrated autonomous replication in mammalian cells.
- Four nuclear polypeptides, including Oct-1 and a p65 protein, specifically bind to the DARC146 sequence.
- A 28 kDa polypeptide from the nuclear matrix also bound to DARC146.
Conclusions:
- The DARC146 fragment contains all necessary signals for DNA synthesis initiation, functioning as a putative replication origin.
- Specific protein-DNA interactions involving Oct-1 and p65 at tandem sites are crucial for DARC146 function.
- A hypothetical model for the DARC146 pre-initiation complex has been proposed.