Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3′ to 5′ translocase, and RecD, a 5′ to 3′ translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair (bp) melting. However, some biochemical evidence suggests that DNA melting of multiple bp may occur separately from single stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity (RP) linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80 bp beyond the RP linkages. This ability requires an ATPase active RecB motor, the RecB “arm” domain and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.

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Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

electrodes. Complementing in vivo genetic assays have been developed and indicate that DNA-mediated signaling between UvrC and other DNAprocessing enzymes containing 4Fe-4S clusters is occurring. Taken together, these results have suggested that UvrC is part of a network of 4Fe-4S proteins that communicate using DNA CT to find lesions and maintain genomic integrity. Additional in vitro and in vivo characterization is underway to understand further the biological implications of the newly-discovered, DNA-mediated redox chemistry of UvrC. 329-Pos Board B109Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation RecBCD is a DNA helicase that functions in repair of double-stranded DNA breaks. RecBCD possesses two SF1 ATPase motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Structure-based models for DNA unwinding by RecBCD propose that RecB and RecD motor translocation is tightly coupled to base pair (bp) melting in that 1 bp is melted as the motors translocate along each single strand by 1 nucleotide for each ATP hydrolyzed. However, there is biochemical evidence to suggest that DNA melting of multiple bp may occur separately from ssDNA translocation and that these two activities may be separable. To test this hypothesis, we designed DNA substrates containing reversals of the phosphodiester backbone polarity (3'-3' and 5'-5' RP linkages) at the same point within the complementary DNA strands that prevent translocation of the canonical RecB and RecD motors. Even though the canonical motors cannot translocate, we observe processive DNA unwinding of at least 80 bp beyond the RP linkages by both RecBCD and RecBC, hence DNA unwinding and ssDNA translocation are separable. This ability is dependent on the previously identified secondary translocase activity residing within RecBC, but does not require RecD. Surprisingly, this ability also requires the nuclease domain of RecB, but not its nuclease activity. These results suggest that RecBCD may couple a double stranded DNA translocase activity to DNA melting and that the nuclease domain plays either an active role in or regulates the helicase activity of RecBCD (supported by NIH GM045948 to TML). 330-Pos Board B110Gene replication and transcription are the core biological processes. These processes are catalyzed by DNA and RNA polymerases. These two types of polymerases show different selectivity on the substrate based on the presence of an extra 2' hydroxyl group on the ribose sugar. DNA polymerases adopt deoxyribose nucleotides (dNTP) while RNA polymerases adopt ribose nucleotides (rNTP). Currently, the general consensus for the sugar discrimination mechanism observed in many nucleotide polymerases is that the highly conserved tyrosine/phenylalanine residues play a critical role. For example, Y416 in RB69 DNA polymerase blocks the bulkier 2' OH on the ribose sugar in rNTP [Yang et al. 2002 Biochem]. However, it is often easy for proteins to avoid such steric clashes by moving the associated residues away which doe...