Thomas G.W. Graham

ParB spreading requires DNA bridging

The parABS system is a widely employed mechanism for plasmid partitioning and chromosome segregation in bacteria. ParB binds to parS sites on plasmids and chromosomes and associates with broad regions of adjacent DNA, a phenomenon known as spreading. Although essential for ParB function, the mechanism of spreading remains poorly understood. Using single-molecule approaches, we discovered that Bacillus subtilis ParB (Spo0J) is able to trap DNA loops. Point mutants in Spo0J that disrupt DNA bridging are defective in spreading and recruitment of structural maintenance of chromosomes (SMC) condensin complexes in vivo. DNA bridging helps to explain how a limited number of Spo0J molecules per parS site (~20) can spread over many kilobases and suggests a mechanism by which ParB proteins could facilitate the loading of SMC complexes. We show that DNA bridging is a property of diverse ParB homologs, suggesting broad evolutionary conservation.

Regulation of the Rev1-pol ζ complex during bypass of a DNA interstrand cross-link.

DNA interstrand cross‐links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL (“approach”), a nucleotide is inserted across from the unhooked lesion (“insertion”), and the leading strand is extended beyond the lesion (“extension”). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site‐specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error‐free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1–pol ζ recruitment requires the Fanconi anemia core complex but not FancI–FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair‐associated mutagenesis.

Two-Stage Synapsis of DNA Ends during Non-homologous End Joining

Repair of DNA double-strand breaks (DSBs) is essential for genomic stability. The most common DSB repair mechanism in human cells, non-homologous end joining (NHEJ), rejoins broken DNA ends by direct ligation. It remains unclear how components of the NHEJ machinery assemble a synaptic complex that bridges DNA ends. Here, we use single-molecule imaging in a vertebrate cell-free extract to show that synapsis of DNA ends occurs in at least two stages that are controlled by different NHEJ factors. DNA ends are initially tethered in a long-range complex whose formation requires the Ku70/80 heterodimer and the DNA-dependent protein kinase catalytic subunit. The ends are then closely aligned, which requires XLF, a non-catalytic function of XRCC4-LIG4, and DNA-PK activity. These results reveal a structural transition in the synaptic complex that governs alignment of DNA ends. Our approach provides a means of studying physiological DNA DSB repair at single-molecule resolution.

A general approach to visualize protein binding and DNA conformation without protein labelling

Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein–DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein–DNA interactions.

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length.

A network of cis and trans interactions is required for ParB spreading

Abstract Most bacteria utilize the highly conserved parABS partitioning system in plasmid and chromosome segregation. This system depends on a DNA-binding protein ParB, which binds specifically to the centromere DNA sequence parS and to adjacent non-specific DNA over multiple kilobases in a phenomenon called spreading. Previous single-molecule experiments in combination with genetic, biochemical and computational studies have argued that ParB spreading requires cooperative interactions between ParB dimers including DNA bridging and possible nearest-neighbor interactions. A recent structure of a ParB homolog co-crystallized with parS revealed that ParB dimers tetramerize to form a higher order nucleoprotein complex. Using this structure as a guide, we systematically ablated a series of proposed intermolecular interactions in the Bacillus subtilis ParB (BsSpo0J) and characterized their effect on spreading using both in vivo and in vitro assays. In particular, we measured DNA compaction mediated by BsSpo0J using a recently developed single-molecule method to simultaneously visualize protein binding on single DNA molecules and changes in DNA conformation without protein labeling. Our results indicate that residues acting as hubs for multiple interactions frequently led to the most severe spreading defects when mutated, and that a network of both cis and trans interactions between ParB dimers is necessary for spreading.

Ensemble and Single-Molecule Analysis of Non-Homologous End Joining in Frog Egg Extracts

Non-homologous end joining (NHEJ) repairs the majority of DNA double-strand breaks in human cells, yet the detailed order of events in this process has remained obscure. Here, we describe how to employ Xenopus laevis egg extract for the study of NHEJ. The egg extract is easy to prepare in large quantities, and it performs efficient end joining that requires the core end joining proteins Ku, DNA-PKcs, XLF, XRCC4, and DNA ligase IV. These factors, along with the rest of the soluble proteome, are present at endogenous concentrations, allowing mechanistic analysis in a system that begins to approximate the complexity of cellular end joining. We describe an ensemble assay that monitors covalent joining of DNA ends and fluorescence assays that detect joining of single pairs of DNA ends. The latter assay discerns at least two discrete intermediates in the bridging of DNA ends.

A single XLF dimer bridges DNA ends during nonhomologous end joining

Nonhomologous end joining (NHEJ) is the primary pathway of DNA double-strand-break repair in vertebrate cells, yet how NHEJ factors assemble a synaptic complex that bridges DNA ends remains unclear. To address the role of XRCC4-like factor (XLF) in synaptic-complex assembly, we used single-molecule fluorescence imaging in Xenopus laevis egg extract, a system that efficiently joins DNA ends. We found that a single XLF dimer binds DNA substrates just before the formation of a ligation-competent synaptic complex between DNA ends. The interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient formation of this synaptic complex. Our results indicate that, in contrast to a model in which filaments of XLF and XRCC4 bridge DNA ends, binding of a single XLF dimer facilitates the assembly of a stoichiometrically well-defined synaptic complex.Single-molecule imaging of nonhomologous end joining in Xenopus egg extract reveals that a single XLF dimer aligns broken DNA ends for ligation.