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Abstract Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme–DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein–protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein–protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading. 

Abstract Protein functional constraints are manifest as superfamily and functional-subgroup conserved residues, and as pairwise correlations. Deep Analysis of Residue Constraints (DARC) aids the visualization of these constraints, characterizes how they correlate with each other and with structure, and estimates statistical significance. This can identify determinants of protein functional specificity, as we illustrate for bacterial DNA clamp loader ATPases. These load ring-shaped sliding clamps onto DNA to keep polymerase attached during replication and contain one δ, three γ, and one δ’ AAA+ subunits semi-circularly arranged in the order δ-γ₁-γ₂-γ₃-δ’. Only γ is active, though both γ and δ’ functionally influence an adjacent γ subunit. DARC identifies, as functionally-congruent features linking allosterically the ATP, DNA, and clamp binding sites: residues distinctive of γ and of γ/δ’ that mutually interact in trans, centered on the catalytic base; several γ/δ’-residues and six γ/δ’-covariant residue pairs within the DNA binding N-termini of helices α2 and α3; and γ/δ’-residues associated with the α2 C-terminus and the clamp-binding loop. Most notable is a trans-acting γ/δ’ hydroxyl group that 99% of other AAA+ proteins lack. Mutation of this hydroxyl to a methyl group impedes clamp binding and opening, DNA binding, and ATP hydrolysis—implying a remarkably clamp-loader-specific function. 

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.