ANALYSIS OF PRIMOSOME ASSEMBLY SITES: STRUCTURAL PROPERTIES AND INTERACTION WITH FACTOR Y
GREENBAUM, JEFFREY HOWARD
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Primosome assembly sites are specific loci of single stranded DNA where seven Escherichia coli encoded proteins associate to form a mobile complex (the primosome) which primes DNA synthesis. These sites are complex structures which share common functions: they elicit the DNA-dependent ATPase of a primosomal protein, replication factor Y, and serve as origins of complementary strand DNA synthesis. However, comparative sequence analysis has revealed little homology among the sites on (CRDIAG)X174 (pas-X) and on the L strand (pas-BL) and H strand (pas-BH) of pBR322. In order to ascertain a common basis for factor Y-DNA recognition, I have employed two types of DNA binding assays, pancreatic DNase footprinting and methylation inhibition/enhancement, to evaluate the physical interaction of factor Y with pas-X, pas-BL, and pas-BH. DNA templates containing pas sequences were treated with pancreatic DNase or dimethyl sulfate in the presence or absence of factor Y. Protection maps, devised by plotting the results of the methylation and footprinting experiments on duplex structures, suggest that selective binding of factor Y is dependent on the structural features of a pas which arise from secondary or tertiary interactions within the single-standed polynucleotide chain. In addition, mutated derivatives of pas-BH have been functionally and structurally analyzed. Under conditions in which they lose the capacity to function as ATPase effectors these DNA templates do not bind factor Y and the DNase cleavage pattern characteristic of this site is perceptibly changed. The results of this study strongly indicate that the structure of this primosome assembly site consists of two hairpins which interact with each other. When the sites of pancreatic DNase attack are plotted on a proposed double hairpin structure, the 5' cleavage sites all map to one duplex while the 3' sites map to the other. The observation that, under factor Y ATPase activating conditions, the 3' hairpin is largely refractory to the action of pancreatic DNase indicates that tertiary interactions between the two duplexes render a portion of the DNA structure inaccessible to the nuclease.