Philip Nevin

Conformational analysis of processivity clamps in solution demonstrates that tertiary structure does not correlate with protein dynamics.

The relationship between protein sequence, structure, and dynamics has been elusive. Here, we report a comprehensive analysis using an in-solution experimental approach to study how the conservation of tertiary structure correlates with protein dynamics. Hydrogen exchange measurements of eight processivity clamp proteins from different species revealed that, despite highly similar three-dimensional structures, clamp proteins display a wide range of dynamic behavior. Differences were apparent both for structurally similar domains within proteins and for corresponding domains of different proteins. Several of the clamps contained regions that underwent local unfolding with different half-lives. We also observed a conserved pattern of alternating dynamics of the α helices lining the inner pore of the clamps as well as a correlation between dynamics and the number of salt bridges in these α helices. Our observations reveal that tertiary structure and dynamics are not directly correlated and that primary structure plays an important role in dynamics.

Selective disruption of the DNA polymerase III α–β complex by the umuD gene products

DNA polymerase III (DNA pol III) efficiently replicates the Escherichia coli genome, but it cannot bypass DNA damage. Instead, translesion synthesis (TLS) DNA polymerases are employed to replicate past damaged DNA; however, the exchange of replicative for TLS polymerases is not understood. The umuD gene products, which are up-regulated during the SOS response, were previously shown to bind to the α, β and ε subunits of DNA pol III. Full-length UmuD inhibits DNA replication and prevents mutagenic TLS, while the cleaved form UmuD′ facilitates mutagenesis. We show that α possesses two UmuD binding sites: at the N-terminus (residues 1–280) and the C-terminus (residues 956–975). The C-terminal site favors UmuD over UmuD′. We also find that UmuD, but not UmuD′, disrupts the α–β complex. We propose that the interaction between α and UmuD contributes to the transition between replicative and TLS polymerases by removing α from the β clamp.

Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III α binding to ssDNA

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.

Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase κ

Y‐family DNA polymerases are specialized to copy damaged DNA, and are associated with increased mutagenesis, owing to their low fidelity. It is believed that the mechanism of nucleotide selection by Y‐family DNA polymerases involves conformational changes preceding nucleotidyl transfer, but there is limited experimental evidence for such structural changes. In particular, nucleotide‐induced conformational changes in bacterial or eukaryotic Y‐family DNA polymerases have, to date, not been extensively characterized. Using hydrogen–deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y‐family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide‐induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N2‐furfuryl‐deoxyguanosine, which is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O6‐methyl‐deoxyguanosine, which is a poor substrate. Our observations suggest that DinB and DNA polymerase κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.

Use of FRET to Study Dynamics of DNA Replication

The DNA replisome is a large protein complex that accurately and efficiently copies an entire genome in every cell cycle. Numerous dynamic interactions must be regulated in order to control this process. Cellular integrity depends on the correct incorporation of each nucleotide and incorrectly incorporated bases must be removed by proofreading to prevent mutations. Lagging strand DNA polymerases and processivity clamps must be continuously recycled. When DNA replication forks encounter damaged DNA, translesion synthesis DNA polymerases replace replicative polymerases to allow bypass of the DNA damage. Forster resonance energy transfer (FRET) has provided key insights into the dynamics of these processes, including how DNA polymerases translocate on DNA, how DNA switches between polymerization and proofreading modes, and the numerous protein–protein and protein–nucleic acid interactions that regulate replication processes. This chapter discusses advances that have been achieved through the application of FRET, including the use of fluorescent nucleotide analogs, to a number of DNA replication processes.