Matthew J. Rossi

Rad54, the motor of homologous recombination

Homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. The invasion produces a cross-stranded structure, known as the Holliday junction. Here, we describe the properties of Rad54, an important and versatile HR protein that is evolutionarily conserved in eukaryotes. Rad54 is a motor protein that translocates along dsDNA and performs several important functions in HR. The current review focuses on the recently identified Rad54 activities which contribute to the late phase of HR, especially the branch migration of Holliday junctions.

A Unitary Anesthetic Binding Site at High Resolution

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABAA receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABAA receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

Cooperation of RAD51 and RAD54 in regression of a model replication fork

DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the ‘template-strand switch’ mechanism. This mechanism involves replication fork regression into a Holliday junction (‘chicken foot structure’), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion.

Rad51 protein stimulates the branch migration activity of Rad54 protein.

The Rad51 and Rad54 proteins play important roles during homologous recombination in eukaryotes. Rad51 forms a nucleoprotein filament on single-stranded DNA and performs the initial steps of double strand break repair. Rad54 belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that Rad54 promotes branch migration of Holliday junctions. Here we find that human Rad51 (hRad51) significantly stimulates the branch migration activity of hRad54. The stimulation appears to be evolutionarily conserved, as yeast Rad51 also stimulates the branch migration activity of yeast Rad54. We further investigated the mechanism of this stimulation. Our results demonstrate that the stimulation of hRad54-promoted branch migration by hRad51 is driven by specific protein-protein interactions, and the active form of the hRad51 filament is more stimulatory than the inactive one. The current results support the hypothesis that the hRad51 conformation state has a strong effect on interaction with hRad54 and ultimately on the function of hRad54 in homologous recombination.

Polarity and Bypass of DNA Heterology during Branch Migration of Holliday Junctions by Human RAD54, BLM, and RECQ1 Proteins

Background: Several proteins catalyze branch migration (BM) of the Holliday junction. Results: RAD54 is a robust BM protein capable of bypassing extensive regions of DNA heterology. RAD54, BLM, and RECQ1 drive BM in the 3′→5′ direction. Conclusion: The displacement strand of joint molecules (JMs) defines the polarity of BM. Significance: BM is mechanistically distinct from helicase activity of DNA translocating proteins. Several proteins have been shown to catalyze branch migration (BM) of the Holliday junction, a key intermediate in DNA repair and recombination. Here, using joint molecules made by human RAD51 or Escherichia coli RecA, we find that the polarity of the displaced ssDNA strand of the joint molecules defines the polarity of BM of RAD54, BLM, RECQ1, and RuvAB. Our results demonstrate that RAD54, BLM, and RECQ1 promote BM preferentially in the 3′→5′ direction, whereas RuvAB drives it in the 5′→3′ direction relative to the displaced ssDNA strand. Our data indicate that the helicase activity of BM proteins does not play a role in the heterology bypass. Thus, RAD54 that lacks helicase activity is more efficient in DNA heterology bypass than BLM or REQ1 helicases. Furthermore, we demonstrate that the BLM helicase and BM activities require different protein stoichiometries, indicating that different complexes, monomers and multimers, respectively, are responsible for these two activities. These results define BM as a mechanistically distinct activity of DNA translocating proteins, which may serve an important function in DNA repair and recombination.

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.

Analyzing the Branch Migration Activities of Eukaryotic Proteins

The Holliday junction is a key intermediate of DNA repair, recombination, and replication. Branch migration of Holliday junctions is a process in which one DNA strand is progressively exchanged for another. Branch migration of Holliday junctions may serve several important functions such as affecting the length of genetic information transferred between homologous chromosomes during meiosis, restarting stalled replication forks, and ensuring the faithful repair of double strand DNA breaks by homologous recombination. Several proteins that promote branch migration of Holliday junctions have been recently identified. These proteins, which function during DNA replication and repair, possess the ability to bind Holliday junctions and other branched DNA structures and drive their branch migration by translocating along DNA in an ATPase-dependent manner. Here, we describe methods employing a wide range of DNA substrates for studying proteins that catalyze branch migration of Holliday junctions.

Correspondence: DNA shape is insufficient to explain binding

Proteins bind DNA through combinations of DNA base and shape recognition1. DNA base recognition refers to a unique arrangement of protein interactions with functional groups on the four DNA bases. Shape recognition refers to protein interactions with specific twists and turns of short stretches of DNA that may deviate from the average three-dimensional shape of B-form DNA. A recent study by Zentner et al.2 characterized the genome-wide binding of S. cerevisiae DNA binding proteins Abf1, Rap1 and Reb1, reporting many thousands of novel, low-scoring binding sites that lacked a consensus motif sequence. The sites were deemed significant because they reportedly possessed a DNA shape that was highly similar to that of the protein’s cognate sites and very different from random sites. We show here that when random sites are processed in precisely the same manner as highand low-scoring sites, including using a 50 bp search window (which by error was not done in Zentner et al.2), the low-scoring sites were no different than random, thereby invalidating the applicable conclusions. Since other analyses on slow sites were interpreted based on these invalid conclusions, we find an overall lack of evidence supporting the conclusion that Abf1, Rap1 and Reb1 predominately read DNA shape to recognize thousands of novel ‘low-scoring’ sites. In Figure 7 of Zentner et al.,2 it was reported that the DNA shape3 of low-scoring sites was on average highly similar to the DNA shapes associated with high-scoring sites and significantly different from random sites. From this result, it was concluded that the favourable DNA shape recognition at low-scoring sites captures transient scanning interactions. The analysis used a 50 bp search space centred over highand low-scoring ChEC-seq (chromatin endogenous cleavage with high-throughput sequencing) peak midpoints, so as to find the best match to a previously published consensus motif4. The P value threshold was set such that up to three mismatches to the consensus motif were allowed. From this, a DNA shape analysis was performed and compared across sites. We repeated the analysis and obtained precisely the same results for highand low-scoring sites (Fig. 1a, red versus blue traces). Most critically, Zentner et al.2 further reported that the same number of random sites, as a negative control, had on average no particular shape property. However, when we repeated this control3, we obtained a shape pattern that was essentially no different from the putative low-scoring sites (Fig. 1a, green versus blue traces). If we excluded the 50 bp search (that is, performed a 1 bp search), then we obtained the pattern reported by Zentner et al.2 (Fig. 1a, black traces). Our reanalysis shows that average DNA shape at low-scoring sites is indistinguishable from random if the best motif is sought equivalently in both data sets. Therefore, this DNA shape analysis3 provides no evidence of shape specificity at putative low-scoring sites, and no evidence that ChEC-seq peaks at low-scoring sites are a product of specific DNA shape recognition. To understand the source of ChEC-seq peaks at putative low-scoring sites, we next investigated the spatial relationship between high-scoring and low-scoring sites. We found that low-scoring sites were physically closer to high-scoring sites in the genome compared to random sites (Fig. 1b). Of the low-scoring sites, B50% were within 250 bp of a high-scoring site (compared to B8% of random sites), indicating that these low-scoring sites generally exist within the same nucleosomedepleted region (NDRs) as high-scoring sites. Consistent with this, Zentner et al.2 reported that AT-rich sequences, which are a well-known property of NDRs, are enriched at low-scoring sites. Since micrococcal nuclease (MNase) preferentially cleaves DNA in NDRs5, we surmise that the ChEC-seq peaks associated with putative low-scoring sites arise mostly from non-specific MNase cleavage events that are near high-scoring sites in accessible chromatin, perhaps after cleavage release from high-scoring sites. This is in accord with their temporally slow appearance (Fig. 3 in Zentner et al.2). Since the high-scoring sites for Abf1, Rap1 and Reb1 do not appreciably overlap, it follows that the positionally linked low-scoring sites would also not overlap, nor overlap with an untargeted MNase-only ‘negative’ control as reported in Zentner et al.2 Figure 4 in Zentner et al.2 reports the distribution of Abf1 X-ChIP-seq (chromatin immunoprecipitation with highthroughput sequencing) peaks around low-scoring sites/motifs. We note a local minimum directly at these motifs, and local maxima B50–100 bp away. These observations are consistent with high-scoring sites, which X-ChIP-seq is measuring, being physically close to but not coincident with putative low-scoring sites. Figure 5 in Zentner et al.2 reports MNase-derived DOI: 10.1038/ncomms15643 OPEN

Genome-wide determinants of sequence-specific DNA binding of general regulatory factors

General regulatory factors (GRFs), such as Reb1, Abf1, Rap1, Mcm1, and Cbf1, positionally organize yeast chromatin through interactions with a core consensus DNA sequence. It is assumed that sequence recognition via direct base readout suffices for specificity and that spurious nonfunctional sites are rendered inaccessible by chromatin. We tested these assumptions through genome-wide mapping of GRFs in vivo and in purified biochemical systems at near-base pair (bp) resolution using several ChIP-exo-based assays. We find that computationally predicted DNA shape features (e.g., minor groove width, helix twist, base roll, and propeller twist) that are not defined by a unique consensus sequence are embedded in the nonunique portions of GRF motifs and contribute critically to sequence-specific binding. This dual source specificity occurs at GRF sites in promoter regions where chromatin organization starts. Outside of promoter regions, strong consensus sites lack the shape component and consequently lack an intrinsic ability to bind cognate GRFs, without regard to influences from chromatin. However, sites having a weak consensus and low intrinsic affinity do exist in these regions but are rendered inaccessible in a chromatin environment. Thus, GRF site-specificity is achieved through integration of favorable DNA sequence and shape readouts in promoter regions and by chromatin-based exclusion from fortuitous weak sites within gene bodies. This study further revealed a severe G/C nucleotide cross-linking selectivity inherent in all formaldehyde-based ChIP assays, which includes ChIP-seq. However, for most tested proteins, G/C selectivity did not appreciably affect binding site detection, although it does place limits on the quantitativeness of occupancy levels.

Reconstituting the Key Steps of the DNA Double-Strand Break Repair In Vitro

Double-stranded DNA breaks (DSB), the most harmful type of DNA lesions, cause cell death and genome instability. Homologous recombination repairs DSB using homologous DNA sequences as templates. Here we describe a set of reactions that lead to reconstitution of the double-stranded DNA break repair process in vitro employing purified human homologous recombination proteins and DNA polymerase η. Reconstitution of critical steps of DSB repair in vitro may help to better understand the mechanisms of recombinational DNA repair and the role of various human homologous recombination proteins in this process.