A combined genetic biochemical approach will be taken to the study of DNA replication in yeast. The way in which these two disciplines will be combined will be called """"""""reverse genetics."""""""" The porteins expected to participate in yeast DNA replication will be purified by specific assays based on previous knowledge, antibodies or oligonucleotides will be prepared and the corresponding genes cloned. Gene replacement and gene distuption techniques will be used to construct mutants useful in establishing whether the individual isolates actually participate in yeast replication or not. Those porteines involved will be used to reconstitutue replication on plasmid DNAS CONTAINING THE ARSl sequence, presumably a chromosomal replicator. For this work, we have cloned the gene and made mutants in the catalytic subunit of the replicative DNA polymerase, DNA polymerase I. Extensive use will be made of the gene and the overproduced protein in characterizing the holoenzyme. We have also isolated a protein that binds specifically to DNAs containing the ARS1 consensus sequence. This protein is a good candidate for a specific initiation protein and this will be investigated by purifying it to homogeneity, preparing antibodies and cloning the gene. The polymerase holoenzyme and the specific initiator protein from the crux of any reconstitution system. Two missing protein links in eukaryotic replication are the helicase and a replicative single-stranded DNA binding protein. Ways to isolate these will be described and once they are obtained reverse genetics will be applied. Several SSBs have already been purified, their genes cloned and intresting mutants prepared. Other proteins or replication will be purified by procedures published by others to be used as reagents: RNase H, primase, topoisomerases etc. Since the gene for primase would be very useful, it will be cloned from a expression library. Ultimately we wish to understand how DNA replicon useage is regulated and we will do this by asking how these proteins and genes are regulated as well as by further studying the DNA sequence, ARSl.
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