ANALYSIS OF THE ROLE OF DNAA RECOGNITION SITE POSITION IN DIRECTING ASSEMBLY OF THE PRE-REPLICATIVE COMPLEX IN ESCHERICHIA COLI
Czerwonka, Christopher Aaron
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Prior to initiating new rounds of DNA replication, all cells assemble pre-replicative complexes (pre-RC) consisting of initiator proteins and regions of DNA termed origins of replication. In the bacterial model system, E. coli, multiple copies of the initiator protein, DnaA,assemble into a complex with the unique chromosomal replication origin, oriC, to produce a pre-RC that unwinds the DNA helix, preparing the origin for the new replication forks. Nine base pair (bp) low affinity DnaA recognition sites are distinctly arrayed in each half of oriC and both arrays are bounded by high affinity recognition sites. Although it is known that DnaA does not interact at low affinity site arrays without assistance from DnaA occupying a proximally positioned high affinity site, it remains unclear why this particular arrangement of high and low affinity sites exists. Furthermore, despite the fact that DnaA is a highly conserved protein among all bacteria, the numbers and arrangements of high and low affinity sites in oriC varies considerably among different bacterial types. In order to understand the reason for this diversity, more information about E. coli oriC geography and its relationship to pre-RC assembly and origin function is required. In the studies reported here, two specific questions about E. coli oriC geography were addressed: 1) are all three high affinity sites required for oriC function, and 2) is the nucleotide spacing between recognition sites an inflexible feature. To answer these questions, mutagenized versions of E. coli oriC on chromosomes were evaluated for function in vivo, and DNA fragments containing different spacing and arrangements of high and low affinity recognition sites were examined by electrophoretic mobility shift assays (EMSA), high resolution DNA footprints, and DNA unwinding assays.When inserted into E. coli as replacements for the wild-type chromosomal origin, mutant versions of oriC lacking any two high affinity recognition sites (R1, R2, or R4) were inactive, but origins carrying only one inactivated high affinity DnaA recognition site retained function no matter which site was altered. This finding contradicts previous reports that the R1siteis essential for oriC function, although loss of either R1 or R4 binding caused reduced origin activity and loss of initiation synchrony.Although it appears that the requirement for high affinity DnaA binding is not stringent for origin activity, a novel phenotype for high affinity site-deficient mutants was identified. Supercoiled oriC DNA is not normally unwound at low levels of DnaA, but spontaneous DNA unwinding of every high affinity binding mutant was detected under these conditions. A model is proposed whereby oriC DNA is normally constrained by DnaA during the cell cycle to prevent spontaneous initiations and constraint requires the cooperative interactions among the distantly spaced DnaA bound to high affinity sites as an origin recognition complex. These interactions, presumably through the proper positioning of domain I-domain I contacts,would generate multiple DNA loops, analogous to the wrapping of DNA around nucleosomes in eukaryotes.Further contraction and stabilization of these loops by additional DnaA occupying arrayed low affinity sites would then produce the torsional stress required to separate DNA strands within the A-T rich DNA Unwinding Element (DUE)at the left side of oriC.Electrophoretic mobility shift assays (EMSA)with DNA fragments containing differently spaced DnaA recognition sites revealed that very small changes in base pair spacing between low-low or high-low affinity recognition sites resulted in dramatic changes in cooperative DnaA binding required for site occupation.Optimal binding at any two sites was detected at 2 bp spacing, but was diminished as single bases were added.Surprisingly, reducing the 2 bp spacing also diminished DnaA binding suggesting that some structural interference exists between adjacent DnaA molecules when they are brought too close together. These observations are consistent with specific positioning of DnaA recognition sites in E. coli oriC for correct origin function and even slight alterations in these positions have an impact. While it remains difficult to predict where DnaA recognition sites will be placed in the replication origin of any particular bacteria type, the knowledge gained from the studies presented here establishes a rule set that include the requirement for the arrangement of high affinity DnaA recognition sites to, at an early stage in the cell cycle, topologically constrain the oriC in an origin recognition complex and the requirement for additional recognition sites that convert the constrained complex into one that imparts sufficient torsional stress to unwind oriC.