Characterization of the sea urchin homologue of the replication factor A 70 kD subunit and the novel interspersed repeat family to which it binds

Citation

Anderson, Roger Francis (1996) Characterization of the sea urchin homologue of the replication factor A 70 kD subunit and the novel interspersed repeat family to which it binds. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/dh99-m567. https://resolver.caltech.edu/CaltechETD:etd-09212006-131348

Abstract

The 5' regulatory region of the Strogylocentrotus purpuratus cytoskeletal actin gene, CyIIIa, contains a 150 bp inverted interspersed repeat. The center of the inverted repeats contains binding sites for a 12 zinc finger DNA binding protein; SpZ12-1. This repeat was also found in the flanking region of the genes for two factors; SpZ2-1 and SpP3A2 which bind to the CyIIIa promoter. The high degree of conservation of the repeats is indicative of potential protein binding sites. Gel-shift assays using sea urchin egg cytoplasmic extracts revealed a second binding activity, on a separate region of the repeat, for a protein which we later identified as the sea urchin homologue of the 70 kD subunit of replication factor A, SpRFA-70.

Replication factor A is a well known single-stranded DNA (ssDNA) binding protein that is required for DNA replication (Kim et al., Mol. Cell. Biol. 12, 3050-3059, 1992). We demonstrate that in addition to its non-specific affinity for ssDNA, SpRFA-70 has a high sequence specific binding affintiy for double-stranded DNA (dsDNA). The equilibrium constant (Keq) for SpRFA-70 binding to ssDNA is 1.7 x 10(10)M-1 and the Keq, for binding to the specific dsDNA site is 1.8 x 10(9)M-1 as determined by protein excess titration. Quantitative gel retardation assays reveal that the SpRFA-70 dsDNA binding activity is two orders of magnitude more prevalent per egg than per whole embryo at 24 hours. These data suggest that SpRFA-70 functions as a regulatory factor predominantly in the egg and early embryo.

A genomic library was screened with short oligo probes to determine the number of sites for the DNA binding factors SpZ12-1 and SpRFA-70. The results of this screen indicate there are ~460 SpRFA-70 sites and ~318 SpZ12-1 sites. These sites match to a consensus sequence by >80%. There are 40-80 instances where these sites occur together. This is 30 fold higher than would be expected by random association. The distance between the two binding sites is also conserved. This arrangement is the same as that which occurs in the CyIIIa regulatory region. That this configuration is so conserved implies a potential regulatory interaction for these two factors.

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