Biology subjects

Albrecht, C. S.

Publications and source records attributed to Albrecht, C. S..

5 recordsLinked to original sources

bioRxiv2025

Functional integration of the bacteriophage T4 DNA replication complex: The multiple roles of the ssDNA binding protein (gp32)

Single-stranded DNA binding protein (gp32) serves as the central regulatory component of the multi-subunit T4 bacteriophage DNA replication system by coordinating the systems three functional sub-assemblies, resulting in phage DNA synthesis in T4-infected E. coli cells at the high speeds ([~]1,000 nts s-1) and the high fidelity (< 1 error per 107 nts) required for genomic function within this cellular eco-system. Gp32 proteins continuously bind to, slide as cooperatively-linked clusters on, and un-bind from transiently exposed single-stranded (ss) DNA templates to carry out their coordinating functions, as well as to protect genomic sequences from nuclease activity and block the formation of interfering secondary structures. The N-terminal domains (NTDs) of gp32 mediate cooperative interactions within ssb clusters, but the roles of the disordered C-terminal domains (CTD) in the nucleation of gp32-ssDNA filaments at ss-dsDNA junctions are less well understood. We here present microsecond-resolved single-molecule Forster resonance energy transfer studies of the initial steps of gp32 assembly on short oligo-deoxythymidine lattices of varying lattice length and polarity near model ss-dsDNA junctions. These data are analyzed to define the molecular steps and related free energy surfaces involved in initiating gp32 cluster formation, which show that the nucleation mechanisms and regulatory interactions driven by gp32 proteins at ss-dsDNA junctions are significantly directed by lattice polarity. We propose a model for the role of the CTDs in orienting gp32 monomers at lattice positions close to ss-dsDNA junctions that suggests how intrinsically disordered CTD domains might facilitate and control non-base-sequence-specific binding in both the nucleation and the dissociation of the gp32-ssDNA filaments involved in phage DNA replication and related processes.

biochemistry↗

bioRxiv2024

Site-specific free energy surface parameters from single-molecule fluorescence measurements of exciton-coupled (iCy3)2 dimer probes positioned at DNA replication fork junctions

Single-stranded (ss) - double-stranded (ds) DNA replication forks and primer-template junctions are important recognition sites for the assembly and function of proteins involved in DNA replication, recombination and repair. DNA breathing - i.e., thermally-induced local fluctuations of the sugar-phosphate backbones and bases - can populate metastable conformational macrostates at positions near such junctions and likely play key roles in the functional interactions of the regulatory proteins that bind at these sites. Recently, Maurer et al. (1) performed polarization-sweep single-molecule fluorescence (PS-SMF) studies on exciton-coupled (iCy3)2 dimer-labeled ss-dsDNA fork constructs, which revealed that the nucleobases and backbones immediately adjacent to the dimer probes undergo conformational fluctuations on time scales ranging from hundreds-of-microseconds to hundreds-of-milliseconds. The local conformations sensed by the dimer probes consist of four quasi-stable macrostates whose populations and dynamics depend on dimer probe position relative to these junctions. Here we present theoretical analyses of these PS-SMF data that quantify the relative stabilities and activation barriers of the free energy surfaces (FESs) of site-specific DNA breathing events at key positions within these junctions. Our results suggest a detailed molecular picture for DNA breathing at these positions, thus providing insights into understanding the molecular mechanisms of the proteins that operate at these sites.

biophysics↗

bioRxiv2024

Two-photon excitation two-dimensional fluorescence spectroscopy (2PE-2DFS) of the fluorescent nucleobase 6-MI

Base stacking is fundamentally important to the stability of double-stranded DNA. However, few experiments can directly probe the local conformations and conformational fluctuations of the DNA bases. Here we report a new spectroscopic approach to study the local conformations of DNA bases using the UV-absorbing fluorescent guanine analogue, 6-methyl isoxanthopterin (6-MI), which can be used as a site-specific probe to label DNA. In these experiments, we apply a two-photon excitation (2PE) approach to two-dimensional fluorescence spectroscopy (2DFS), which is a fluorescence-detected nonlinear Fourier transform spectroscopy. In 2DFS, a repeating sequence of four collinear laser pulses (with center wavelength [~] 675 nm and relative phases swept at radio frequencies) is used to excite the lowest energy electronic-vibrational (vibronic) transitions of 6-MI (with center wavelength [~] 340 nm). The ensuing low flux fluorescence is phase-synchronously detected at the level of individual photons and as a function of inter-pulse delay. The 2PE transition pathways that give rise to electronically excited state populations include optical coherences between electronic ground and excited states and non-resonant (one-photon-excited) virtual states. Our results indicate that 2PE-2DFS experiments can provide information about the electronic-vibrational spectrum of the 6-MI monomer, in addition to the conformation-dependent exciton coupling between adjacent 6-MI monomers within a (6-MI)2 dimer. In principle, this approach can be used to determine the local base-stacking conformations of (6-MI)2 dimer-substituted DNA constructs.

biophysics↗

bioRxiv2023

Studies of DNA "breathing" by polarization-sweep single-molecule fluorescence microscopy of exciton-coupled (iCy3)2 dimer-labeled DNA fork constructs

Local fluctuations of the sugar-phosphate backbones and bases of DNA (often called DNA breathing) play a variety of critical roles in controlling the functional interactions of the DNA genome with the protein complexes that regulate it. Here we present a single-molecule fluorescence method that we have used to measure and characterize such conformational fluctuations at and near biologically important positions in model DNA replication fork constructs labeled with exciton-coupled cyanine [(iCy3)2] dimer probes. Previous work has shown that the constructs that we test here exhibit a broad range of spectral properties at the ensemble level, and these differences can be structurally and dynamically interpreted using our present methodology at the single-molecule level. The (iCy3)2 dimer has one symmetric (+) and one anti-symmetric (-) exciton with respective transition dipole moments oriented perpendicular to one another. We excite single molecule samples using a continuous-wave linearly polarized laser with polarization direction continuously rotated at the frequency 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and the anti-symmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and conformational interconversion dynamics of the (iCy3)2 probe. We find that at most construct positions that we examined the (iCy3)2 dimer-labeled DNA fork constructs can adopt four topologically distinct conformational macrostates. These results suggest that in addition to observing DNA breathing at and near ss-dsDNA junctions, our new methodology should be useful to determine which of these pre-existing macrostates are recognized by, bind to, and are stabilized by various genome regulatory proteins.

biophysics↗

bioRxiv2021

Sub-millisecond conformational transitions of short single-stranded DNA lattices by photon correlation single-molecule FRET

Thermally-driven conformational fluctuations (or breathing) of DNA plays important roles in the function and regulation of the macromolecular machinery of genome expression. Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here we use microsecond-resolved single-molecule Forster Resonance Energy Transfer (smFRET) experiments to investigate the backbone fluctuations of short oligothymidine [oligo(dT)n] templates within DNA constructs that can also serve as models for ss-dsDNA junctions. Such junctions, as well as the attached ssDNA sequences, are involved in the binding of ssDNA binding (ssb) proteins that control and integrate the mechanisms of DNA replication complexes. We have used these data to determine multi-order time-correlation functions (TCFs) and probability distribution functions (PDFs) that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs inter-convert, on sub-millisecond time-scales, between three macrostates with distinctly different end-to-end distances. These are: (i) a compact macrostate that represents the dominant species at equilibrium; (ii) a partially extended macrostate that exists as a minority species; and (iii) a highly extended macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA associating proteins. Significance StatementThe genetic information of living organisms is encoded as sequences of nucleic acid bases in DNA, and is protected by the thermodynamically stable secondary structure of the Watson-Crick double helix. The processing and manipulation of gene sequences by macromolecular machines requires that stable segments of duplex DNA be disrupted, and that single-stranded (ss) DNA templates be transiently exposed to the binding sites of DNA associating proteins within the cellular environment. Here we elucidate some of the defining features that control the stability and dynamics of ssDNA secondary structure, using time-resolved methods to detect the presence of transient unstable conformations. Understanding the nature of these instabilities is central to elucidating the mechanisms by which ssDNA templates facilitate protein binding and function.

biophysics↗