Hop2-Mnd1 functions as a DNA sequence fidelity switch in Dmc1-mediated DNA recombination.

Homologous recombination during meiosis is critical for chromosome segregation and also gives rise to genetic diversity. Genetic exchange between homologous chromosomes during meiosis is mediated by the recombinase Dmc1, which is capable of recombining DNA sequences with mismatches. The Hop2-Mnd1 complex mediates Dmc1 activity.

Protocol for detecting homologous recombination activity in cancer cells by adenovirus-based functional assay.

Cancer cells with homologous recombination deficiency (HRD) exhibit a distinctive vulnerability to poly(ADP-ribose) polymerase inhibitors (PARPis). To address the limitations of existing methodologies incapable of providing real-time insights into homologous recombination (HR) status, we present an adenovirus-based functional assay designed to quantify cellular HR activity. Here, we delineate the step-by-step procedure for producing the adenovirus harboring an HR reporter, processing primary cells, and assessing HR activity in primary ovarian cancer cells.

Mei5-Sae3 stabilizes Dmc1 nucleating clusters for efficient Dmc1 assembly on RPA-coated single-stranded DNA.

Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5-Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5-Sae3 are interdependent in their colocalization on the chromosomes.

Biochemical characterization of the meiosis-essential yet evolutionarily divergent topoisomerase VIB-like protein MTOPVIB from Arabidopsis thaliana.

Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage.

CaMKK2 and CHK1 phosphorylate human STN1 in response to replication stress to protect stalled forks from aberrant resection.

Keeping replication fork stable is essential for safeguarding genome integrity; hence, its protection is highly regulated. The CTC1-STN1-TEN1 (CST) complex protects stalled forks from aberrant MRE11-mediated nascent strand DNA degradation (NSD). However, the activation mechanism for CST at forks is unknown.

An activity-based functional test for identifying homologous recombination deficiencies across cancer types in real time.

Homologous recombination (HR)-mediated DNA repair is a prerequisite for maintaining genome stability. Cancer cells displaying HR deficiency (HRD) are selectively eliminated by poly(ADP-ribose) polymerase inhibitors (PARPis). To date, sequencing of HR-associated genes and analyzing genome instability have been used as clinical predictions for PARPi therapy.

RAD51 paralogs synergize with RAD51 to protect reversed forks from cellular nucleases.

Fork reversal is a conserved mechanism to prevent stalled replication forks from collapsing. Formation and protection of reversed forks are two crucial steps in ensuring fork integrity and stability. Five RAD51 paralogs, namely, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which share sequence and structural similarity to the recombinase RAD51, play poorly defined mechanistic roles in these processes.

A RAD51-ADP double filament structure unveils the mechanism of filament dynamics in homologous recombination.

ATP-dependent RAD51 recombinases play an essential role in eukaryotic homologous recombination by catalyzing a four-step process: 1) formation of a RAD51 single-filament assembly on ssDNA in the presence of ATP, 2) complementary DNA strand-exchange, 3) ATP hydrolysis transforming the RAD51 filament into an ADP-bound disassembly-competent state, and 4) RAD51 disassembly to provide access for DNA repairing enzymes. Of these steps, filament dynamics between the ATP- and ADP-bound states, and the RAD51 disassembly mechanism, are poorly understood due to the lack of near-atomic-resolution information of the ADP-bound RAD51-DNA filament structure. We report the cryo-EM structure of ADP-bound RAD51-DNA filaments at 3.

Flap endonuclease Rad27 cleaves the RNA of R-loop structures to suppress telomere recombination.

The long non-coding telomeric RNA transcript TERRA, in the form of an RNA-DNA duplex, regulates telomere recombination. In a screen for nucleases that affects telomere recombination, mutations in DNA2, EXO1, MRE11 and SAE2 cause severe delay in type II survivor formation, indicating that type II telomere recombination is mediated through a mechanism similar to repairing double-strand breaks. On the other hand, mutation in RAD27 results in early formation of type II recombination, suggesting that RAD27 acts as a negative regulator in telomere recombination.

PARP1 recruits DNA translocases to restrain DNA replication and facilitate DNA repair.

Replication fork reversal which restrains DNA replication progression is an important protective mechanism in response to replication stress. PARP1 is recruited to stalled forks to restrain DNA replication. However, PARP1 has no helicase activity, and the mechanism through which PARP1 participates in DNA replication restraint remains unclear.

Comparison of ATP-binding pockets and discovery of homologous recombination inhibitors.

The ATP binding sites of many enzymes are structurally related, which complicates their development as therapeutic targets. In this work, we explore a diverse set of ATPases and compare their ATP binding pockets using different strategies, including direct and indirect structural methods, in search of pockets attractive for drug discovery. We pursue different direct and indirect structural strategies, as well as ligandability assessments to help guide target selection.

Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation.

Background: RAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear.

Crosstalk between CST and RPA regulates RAD51 activity during replication stress.

Replication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51.

DSB-3 reveals conservation and divergence among protein complexes promoting meiotic double-strand breaks.

Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: Recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved.

Detecting Selection on Segregating Gene Duplicates in a Population.

Gene duplication is a fundamental process that has the potential to drive phenotypic differences between populations and species. While evolutionarily neutral changes have the potential to affect phenotypes, detecting selection acting on gene duplicates can uncover cases of adaptive diversification. Existing methods to detect selection on duplicates work mostly inter-specifically and are based upon selection on coding sequence changes, here we present a method to detect selection directly on a copy number variant segregating in a population.

Moderate prenatal alcohol exposure increases total length of L1-expressing axons in E15.5 mice.

Public health campaigns broadcast the link between heavy alcohol consumption during pregnancy and physical, cognitive, and behavioral birth defects; however, they appear less effective in deterring moderate consumption prevalent in women who are pregnant or of childbearing age. The incidence of mild Fetal Alcohol Spectrum Disorders (FASD) is likely underestimated because the affected individuals lack physical signs such as retarded growth and facial dysmorphology and cognitive/behavioral deficits are not commonly detected until late childhood. Sensory information processing is distorted in FASD, but alcohol's effects on the development of axons that mediate these functions are not widely investigated.

Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast () Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity.

Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance.

Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA.

Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1-STN1-TEN1) proteins, which form a single-stranded DNA-binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent-strand degradation at reversed forks and ssDNA accumulation.

Detecting Signatures of Positive Selection against a Backdrop of Compensatory Processes.

There are known limitations in methods of detecting positive selection. Common methods do not enable differentiation between positive selection and compensatory covariation, a major limitation. Further, the traditional method of calculating the ratio of nonsynonymous to synonymous substitutions (dN/dS) does not take into account the 3D structure of biomacromolecules nor differences between amino acids.

Microcephaly family protein MCPH1 stabilizes RAD51 filaments.

Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2-RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1.

Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase.

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step.

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes.

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. , many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events.

Promotion of homology-directed DNA repair by polyamines.

Polyamines, often elevated in cancer cells, have been shown to promote cell growth and proliferation. Whether polyamines regulate other cell functions remains unclear. Here, we explore whether and how polyamines affect genome integrity.

Recombination-independent rapid convergent evolution of the gastric pathogen Helicobacter pylori.

Background: Helicobacter pylori is a human stomach pathogen, naturally-competent for DNA uptake, and prone to homologous recombination. Extensive homoplasy (i.e.