Purification, molecular cloning, and characterization of a DNA replication enhancer protein, OBF1

Thesis/Dissertation · Wed Jan 01 04:00:00 EST 1992

OSTI ID:7201072

Francesconi, S C

In order to biochemically understand the events which occur during eukaryotic DNA replication, it is necessary to resolve and reconstitute the reaction in vitro. This thesis describes the isolation, molecular cloning, and characterization of a protein which has a role in enhancing the initiation of replication from chromosomal origins in the yeast Saccharomyces cerevisiae. Origin Binding Factor 1 (OBF1) was discovered and purified based on its ability to specifically bind DNA associated with yeast origins of replication. Antibodies, prepared against the pure protein, were used to clone the OBF1 gene from a yeast genomic expression library in [lambda]gt11. The gene encodes a protein of 731 amino acids, with a calculated molecular weight of 81,743 daltons. However, when subjected to electrophoresis on SDS-PAGE, the OBF1 protein migrated heterogeneously as at least five polypeptides ranging in size between 123 kDa-130 kDa. By growing yeast cultures in a medium containing [sup 32]P-ortho-phosphate, I demonstrated that the protein in multiply phosphorylated at serine and threonine residues, which may explain its heterologous migration in SDS-polyacrylamide gels. Five major sites of phosphorylation have been identified and mapped to discrete regions within the OBF1 primary sequence. Analysis of OBF1 throughout the cell cycle revealed that the phosphorylation state of two sites vary; while one site is phosphorylated only during mitosis, the other is only phosphorylated during S-phase. The cell cycle specific phosphorylation suggests a regulation mechanism for OBF1 activities in DNA replication and transcription.

• Research Organization: Connecticut Univ., Storrs, CT (United States)
• OSTI ID: 7201072
• Country of Publication: United States
• Language: English

Related Subjects

550300* -- Cytology
550400 -- Genetics
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CHEMICAL REACTIONS
CLONING
DNA REPLICATION
EUMYCOTA
FUNGI
IN VITRO
KINETICS
MICROORGANISMS
MOLECULAR BIOLOGY
NUCLEIC ACID REPLICATION
ORGANIC COMPOUNDS
PHOSPHORYLATION
PLANTS
PROTEINS
PURIFICATION
REACTION KINETICS
SACCHAROMYCES
SACCHAROMYCES CEREVISIAE
YEASTS