MutL Activates UvrD by Interaction Between the MutL C-terminal Domain and the UvrD 2B Domain.

UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3'-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.

Subunit Communication within Dimeric SF1 DNA Helicases.

Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with ∼10-fold lower rates and lower processivities than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.

Stimulation of ATP Hydrolysis by ssDNA Provides the Necessary Mechanochemical Energy for G4 Unfolding.

The G-quadruplex (G4) is a distinct geometric and electrophysical structure compared to classical double-stranded DNA, and its stability can impede essential cellular processes such as replication, transcription, and translation. This study focuses on the BsPif1 helicase, revealing its ability to bind independently to both single-stranded DNA (ssDNA) and G4 structures. The unfolding activity of BsPif1 on G4 relies on the presence of a single tail chain, and the covalent continuity between the single tail chain and the G4's main chain is necessary for efficient G4 unwinding. This suggests that ATP hydrolysis-driven ssDNA translocation exerts a pull force on G4 unwinding. Molecular dynamics simulations identified a specific region within BsPif1 that contains five crucial amino acid sites responsible for G4 binding and unwinding. A "molecular wire stripper" model is proposed to explain BsPif1's mechanism of G4 unwinding. These findings provide a new theoretical foundation for further exploration of the G4 development mechanism in Pif1 family helicases.

E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity.

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecBΔNucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecBD1080ACD), which retains the nuclease domain, showed no change in ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. RecBΔNucCD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecBΔNucCD. Surprisingly, significant pauses in DNA unwinding occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, possibly allosterically and that RecBΔNucCD may mimic a post-chi state of RecBCD.

Joint Efforts of Replicative Helicase and SSB Ensure Inherent Replicative Tolerance of G-Quadruplex.

G-quadruplex (G4) is a four-stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single-molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging-strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single-stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading-strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

Harmonin homology domain-mediated interaction of RTEL1 helicase with RPA and DNA provides insights into its recruitment to DNA repair sites.

The regulator of telomere elongation helicase 1 (RTEL1) plays roles in telomere DNA maintenance, DNA repair, and genome stability by dismantling D-loops and unwinding G-quadruplex structures. RTEL1 comprises a helicase domain, two tandem harmonin homology domains 1&2 (HHD1 and HHD2), and a Zn2+-binding RING domain. In vitro D-loop disassembly by RTEL1 is enhanced in the presence of replication protein A (RPA). However, the mechanism of RTEL1 recruitment at non-telomeric D-loops remains unknown. In this study, we have unravelled a direct physical interaction between RTEL1 and RPA. Under DNA damage conditions, we showed that RTEL1 and RPA colocalise in the cell. Coimmunoprecipitation showed that RTEL1 and RPA interact, and the deletion of HHDs of RTEL1 significantly reduced this interaction. NMR chemical shift perturbations (CSPs) showed that RPA uses its 32C domain to interact with the HHD2 of RTEL1. Interestingly, HHD2 also interacted with DNA in the in vitro experiments. HHD2 structure was determined using X-ray crystallography, and NMR CSPs mapping revealed that both RPA 32C and DNA competitively bind to HHD2 on an overlapping surface. These results establish novel roles of accessory HHDs in RTEL1's functions and provide mechanistic insights into the RPA-mediated recruitment of RTEL1 to DNA repair sites.

Using a Function-First "Scout Fragment"-Based Approach to Develop Allosteric Covalent Inhibitors of Conformationally Dynamic Helicase Mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic "scout fragments", which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.

Chromatin gatekeeper and modifier CHD proteins in development, and in autism and other neurological disorders.

Chromatin, a protein-DNA complex, is a dynamic structure that stores genetic information within the nucleus and responds to molecular/cellular changes in its structure, providing conditional access to the genetic machinery. ATP-dependent chromatin modifiers regulate access of transcription factors and RNA polymerases to DNA by either "opening" or "closing" the structure of chromatin, and its aberrant regulation leads to a variety of neurodevelopmental disorders. The chromodomain helicase DNA-binding (CHD) proteins are ATP-dependent chromatin modifiers involved in the organization of chromatin structure, act as gatekeepers of genomic access, and deposit histone variants required for gene regulation. In this review, we first discuss the structural and functional domains of the CHD proteins, and their binding sites, and phosphorylation, acetylation, and methylation sites. The conservation of important amino acids in SWItch/sucrose non-fermenting (SWI/SNF) domains, and their protein and mRNA tissue expression profiles are discussed. Next, we convey the important binding partners of CHD proteins, their protein complexes and activities, and their involvements in epigenetic regulation. We also show the ChIP-seq binding dynamics for CHD1, CHD2, CHD4, and CHD7 proteins at promoter regions of histone genes, as well as several genes that are critical for neurodevelopment. The role of CHD proteins in development is also discussed. Finally, this review provides information about CHD protein mutations reported in autism and neurodevelopmental disorders, and their pathogenicity. Overall, this review provides information on the progress of research into CHD proteins, their structural and functional domains, epigenetics, and their role in stem cell, development, and neurological disorders.

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain.

BACKGROUND: The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). RESULTS: Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. CONCLUSIONS: We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.

Molecular wrench activity of DNA helicases: Keys to modulation of rapid kinetics in DNA repair.

DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP-stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a "molecular wrench" mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP-stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox-active DNA monolayers. StXPB2 ATP-stimulated activity is concentration-independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration-dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB-Bax1 inhibits rapid activity, whereas the StXPB2-Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair.

Identification and characterization of RuvBL DNA helicase genes for tolerance against abiotic stresses in bread wheat (Triticum aestivum L.) and related species.

Recombination UVB (sensitivity) like (RuvBL) helicase genes represent a conserved family of genes, which are known to be involved in providing tolerance against abiotic stresses like heat and drought. We identified nine wheat RuvBL genes, one each on nine different chromosomes, belonging to homoeologous groups 2, 3, and 4. The lengths of genes ranged from 1647 to 2197 bp and exhibited synteny with corresponding genes in related species including Ae. tauschii, Z. mays, O. sativa, H. vulgare, and B. distachyon. The gene sequences were associated with regulatory cis-elements and transposable elements. Two genes, namely TaRuvBL1a-4A and TaRuvBL1a-4B, also carried targets for a widely known miRNA, tae-miR164. Gene ontology revealed that these genes were closely associated with ATP-dependent formation of histone acetyltransferase complex. Analysis of the structure and function of RuvBL proteins revealed that the proteins were localized mainly in the cytoplasm. A representative gene, namely TaRuvBL1a-4A, was also shown to be involved in protein-protein interactions with ten other proteins. On the basis of phylogeny, RuvBL proteins were placed in two sub-divisions, namely RuvBL1 and RuvBL2, which were further classified into clusters and sub-clusters. In silico studies suggested that these genes were differentially expressed under heat/drought. The qRT-PCR analysis confirmed that expression of TaRuvBL genes differed among wheat cultivars, which differed in the level of thermotolerance. The present study advances our understanding of the biological role of wheat RuvBL genes and should help in planning future studies on RuvBL genes in wheat including use of RuvBL genes in breeding thermotolerant wheat cultivars.

Activity, substrate preference and structure of the HsMCM8/9 helicase.

The minichromosomal maintenance proteins, MCM8 and MCM9, are more recent evolutionary additions to the MCM family, only cooccurring in selected higher eukaryotes. Mutations in these genes are directly linked to ovarian insufficiency, infertility, and several cancers. MCM8/9 appears to have ancillary roles in fork progression and recombination of broken replication forks. However, the biochemical activity, specificities and structures have not been adequately illustrated, making mechanistic determination difficult. Here, we show that human MCM8/9 (HsMCM8/9) is an ATP dependent DNA helicase that unwinds fork DNA substrates with a 3'-5' polarity. High affinity ssDNA binding occurs in the presence of nucleoside triphosphates, while ATP hydrolysis weakens the interaction with DNA. The cryo-EM structure of the HsMCM8/9 heterohexamer was solved at 4.3 Å revealing a trimer of heterodimer configuration with two types of interfacial AAA+ nucleotide binding sites that become more organized upon binding ADP. Local refinements of the N or C-terminal domains (NTD or CTD) improved the resolution to 3.9 or 4.1 Å, respectively, and shows a large displacement in the CTD. Changes in AAA+ CTD upon nucleotide binding and a large swing between the NTD and CTD likely implies that MCM8/9 utilizes a sequential subunit translocation mechanism for DNA unwinding.

Global Discovery of Covalent Modulators of Ribonucleoprotein Granules.

Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response, viral infection, and the tumor microenvironment. While proteomic and transcriptomic investigations of SGs and PBs have provided insights into molecular composition, chemical tools to probe and modulate RNP granules remain lacking. Herein, we combine an immunofluorescence (IF)-based phenotypic screen with chemoproteomics to identify sulfonyl-triazoles (SuTEx) capable of preventing or inducing SG and PB formation through liganding of tyrosine (Tyr) and lysine (Lys) sites in stressed cells. Liganded sites were enriched for RNA-binding and protein-protein interaction (PPI) domains, including several sites found in RNP granule-forming proteins. Among these, we functionally validate G3BP1 Y40, located in the NTF2 dimerization domain, as a ligandable site that can disrupt arsenite-induced SG formation in cells. In summary, we present a chemical strategy for the systematic discovery of condensate-modulating covalent small molecules.

Insights into the Dynamics and Helicase Activity of RecD2 of Deinococcus radiodurans during DNA Repair: A Single-Molecule Perspective.

While the double helix is the most stable conformation of DNA inside cells, its transient unwinding and subsequent partial separation of the two complementary strands yields an intermediate single-stranded DNA (ssDNA). The ssDNA is involved in all major DNA transactions such as replication, transcription, recombination, and repair. The process of DNA unwinding and translocation is shouldered by helicases that transduce the chemical energy derived from nucleotide triphosphate (NTP) hydrolysis to mechanical energy and utilize it to destabilize hydrogen bonds between complementary base pairs. Consequently, a comprehensive understanding of the molecular mechanisms of these enzymes is essential. In the last few decades, a combination of single-molecule techniques (force-based manipulation and visualization) have been employed to study helicase action. These approaches have allowed researchers to study the single helicase-DNA complex in real-time and the free energy landscape of their interaction together with the detection of conformational intermediates and molecular heterogeneity during the course of helicase action. Furthermore, the unique ability of these techniques to resolve helicase motion at nanometer and millisecond spatial and temporal resolutions, respectively, provided surprising insights into their mechanism of action. This perspective outlines the contribution of single-molecule methods in deciphering molecular details of helicase activities. It also exemplifies how each technique was or can be used to study the helicase action of RecD2 in recombination DNA repair.

Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a ß-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the ß-bulge loop is required for supercoiling activity. It has previously been shown that the ß-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a ß-bulge loop as a minimal latch is reported. It is shown that the ß-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.

Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.

Biochemical characterisation of Mer3 helicase interactions and the protection of meiotic recombination intermediates.

Crossing over between homologs is critical for the stable segregation of chromosomes during the first meiotic division. Saccharomyces cerevisiae Mer3 (HFM1 in mammals) is a SF2 helicase and member of the ZMM group of proteins, that facilitates the formation of the majority of crossovers during meiosis. Here, we describe the structural organisation of Mer3 and using AlphaFold modelling and XL-MS we further characterise the previously described interaction with Mlh1-Mlh2. We find that Mer3 also forms a previously undescribed complex with the recombination regulating factors Top3 and Rmi1 and that this interaction is competitive with Sgs1BLM helicase. Using in vitro reconstituted D-loop assays we show that Mer3 inhibits the anti-recombination activity of Sgs1 helicase, but only in the presence of Dmc1. Thus we provide a mechanism whereby Mer3 interacts with a network of proteins to protect Dmc1 derived D-loops from dissolution.

The LH-DH module of bacterial replicative helicases is the common binding site for DciA and other helicase loaders.

During the initiation step of bacterial genome replication, replicative helicases depend on specialized proteins for their loading onto oriC. DnaC and DnaI were the first loaders to be characterized. However, most bacteria do not contain any of these genes, which are domesticated phage elements that have replaced the ancestral and unrelated loader gene dciA several times during evolution. To understand how DciA assists the loading of DnaB, the crystal structure of the complex from Vibrio cholerae was determined, in which two VcDciA molecules interact with a dimer of VcDnaB without changing its canonical structure. The data showed that the VcDciA binding site on VcDnaB is the conserved module formed by the linker helix LH of one monomer and the determinant helix DH of the second monomer. Interestingly, DnaC from Escherichia coli also targets this module onto EcDnaB. Thanks to their common target site, it was shown that VcDciA and EcDnaC could be functionally interchanged in vitro despite sharing no structural similarity. This represents a milestone in understanding the mechanism employed by phage helicase loaders to hijack bacterial replicative helicases during evolution.

Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing.

DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme. Using novel HelQ mutant proteins we identify that inhibition of Pol δ is independent of DNA binding, and maps to a 70 amino acid intrinsically disordered region of HelQ. Pol δ and its POLD3 subunit robustly stimulated DNA single-strand annealing by HelQ, and POLD3 and HelQ interact physically via the intrinsically disordered HelQ region. This data, and inability of HelQ to inhibit DNA synthesis by the POLD1 catalytic subunit of Pol δ, reveal a mechanism for limiting DNA synthesis and promoting DNA strand annealing during human DNA break repair, which centres on POLD3.

Rad5 HIRAN domain: Structural insights into its interaction with ssDNA through molecular modeling approaches.

The Rad5 protein is an SWI/SNF family ubiquitin ligase that contains an N-terminal HIRAN domain and a RING C3HC4 motif. The HIRAN domain is critical for recognition of the stalled replication fork during the replication process and acts as a sensor to initiate the damaged DNA checkpoint. It is a conserved domain widely distributed in eukaryotic organisms and is present in several DNA-binding proteins from all kingdoms. Here we showed that distant species have important differences in key residues that affect affinity for ssDNA. Based on these findings, we hypothesized that different HIRAN domains might affect fork reversal and translesion synthesis through different metabolic processes. To address this question, we predicted the tertiary structure of both yeast and human HIRAN domains using molecular modeling. Structural dynamics experiments showed that the yeast HIRAN domain exhibited higher structural denaturation than its human homolog, although both domains became stable in the presence of ssDNA. Analysis of atomic contacts revealed that a greater number of interactions between the ssDNA nucleotides and the Rad5 domain are electrostatic. Taken together, these results provide new insights into the molecular mechanism of the HIRAN domain of Rad5 and may guide us to further elucidate differences in the ancient eukaryotes HIRAN sequences and their DNA affinity.Communicated by Ramaswamy H. Sarma.