Nasib K. Maluf

5′‐Single‐stranded/duplex DNA junctions are loading sites for E. coli UvrD translocase

Escherichia coli UvrD is a 3′–5′ superfamily 1A helicase/translocase involved in a variety of DNA metabolic processes. UvrD can function either as a helicase or only as an single‐stranded DNA (ssDNA) translocase. The switch between these activities is controlled in vitro by the UvrD oligomeric state; a monomer has ssDNA translocase activity, whereas at least a dimer is needed for helicase activity. Although a 3′‐ssDNA partial duplex provides a high‐affinity site for a UvrD monomer, here we show that a monomer also binds with specificity to DNA junctions possessing a 5′‐ssDNA flanking region and can initiate translocation from this site. Thus, a 5′‐ss–duplex DNA junction can serve as a high‐affinity loading site for the monomeric UvrD translocase, whereas a 3′‐ss–duplex DNA junction inhibits both translocase and helicase activity of the UvrD monomer. Furthermore, the 2B subdomain of UvrD is important for this junction specificity. This highlights a separation of helicase and translocase function for UvrD and suggests that a monomeric UvrD translocase can be loaded at a 5′‐ssDNA junction when translocation activity alone is needed.

Cooperative heteroassembly of the adenoviral L4-22K and IVa2 proteins onto the viral packaging sequence DNA.

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

Self-Association of the Adenoviral L4-22K Protein

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus that causes infections of the respiratory tract, urinary tract, and gastrointestinal tract. Assembly of virus particles requires condensation and encapsidation of the linear viral genome. This process requires sequence specific binding of two viral proteins, called IVa2 and L4-22K, to a conserved sequence located at the left end of the viral genome, called the packaging sequence (PS). IVa2 and an alternatively spliced form of L4-22K, called L4-33K, also function as transcriptional activators of the major late promoter (MLP), which encodes viral structural and core proteins. IVa2 and L4-33K bind to identical conserved DNA sequences downstream of the MLP, called the downstream element (DE), to activate transcription. To begin to dissect how the IVa2, L4-22K, and L4-33K proteins simultaneously function as transcriptional activators and DNA packaging proteins, we need to understand the thermodynamics of assembly of these proteins on DNA that contains the PS as well as the DE. Toward this end, we have characterized the self-assembly properties of highly purified, recombinant L4-22K protein. We show that L4-22K reversibly assembles into higher-order structures according to an indefinite, isodesmic assembly scheme. We show that the smallest polymerizing unit is likely the L4-22K monomer (s(20,w) = 2.16 ± 0.04 S) and that the monomer assembles with itself and/or other aggregates with an equilibrium association constant, L, of 112 (102, 124) μM(-1) (0.1 M NaCl, pH 7, 25 °C). A mechanistic consequence of an isodesmic, indefinite assembly process is that the free concentration of the smallest polymerizing unit cannot exceed 1/L. We discuss the implications of this observation with respect to the thermodynamics of assembly of L4-22K and IVa2 on the PS.