Werner syndrome (WS) is an autosomal recessive disease caused by loss of function of WRN. WS is a segmental progeroid disease and shows early onset or increased frequency of many characteristics of normal aging. WRN possesses helicase, annealing, strand exchange, and exonuclease activities and acts on a variety of DNA substrates, even complex replication and recombination intermediates. Here, we review the genetics, biochemistry, and probably physiological functions of the WRN protein. Although its precise role is unclear, evidence suggests WRN plays a role in pathways that respond to replication stress and maintain genome stability particularly in telomeric regions.

Homologous recombination deficiency (HRD) score is a reliable indicator of genomic instability. The significance of HRD in nasopharyngeal carcinoma (NPC), particularly its influence on prognosis and the immune microenvironment, has yet to be adequately explored. Understanding HRD status comprehensively can offer valuable insights for guiding precision treatment. We utilised three cohorts to investigate HRD status in NPC: the Zhujiang cohort from local collection and the Hong Kong (SRA288429) and Singapore (SRP035573) cohorts from public datasets. The GATK (genome analysis toolkit) best practice process was employed to investigate germline and somatic BRCA1/2 mutations and various bioinformatics tools and algorithms to examine the association between HRD status and clinical molecular characteristics. We found that individuals with a negative HRD status (no-HRD) exhibited a higher risk of recurrence [hazard ratio (HR), 1.43; 95% confidence interval (CI), 2.03-333.76; p = 0.012] in the Zhujiang cohort, whereas, in the Singapore cohort, they experienced a higher risk of mortality (HR, 26.04; 95% CI, 1.43-34.21; p = 0.016) compared with those in the HRD group. In vitro experiments demonstrated that NPC cells with BRCA1 knockdown exhibit heightened sensitivity to chemoradiotherapy. Furthermore, the HRD group showed significantly higher tumour mutational burden and tumour neoantigen burden levels than the no-HRD group. Immune infiltration analysis indicated that HRD tissues tend to have a non-inflamed tumour microenvironment. In conclusion, patients with HRD exhibit a comparatively favourable prognosis in NPC, possibly associated with a non-inflammatory immune microenvironment. These findings have positive implications for treatment stratification, enabling the selection of more precise and effective therapeutic approaches and aiding in the prediction of treatment response and prognosis to a certain extent.

The repair of DNA double-strand breaks (DSBs) through alternative non-homologous end-joining (alt-NHEJ) pathway significantly contributes to genetic instability. However, the mechanism governing alt-NHEJ pathway choice, particularly its association with DSB complexity, remains elusive due to the absence of a suitable reporter system. In this study, we established a unique Escherichia coli reporter system for detecting complex DSB-initiated alternative end-joining (A-EJ), an alt-NHEJ-like pathway. By utilizing various types of ionizing radiation to generate DSBs with varying degrees of complexity, we discovered that high complexity of DSBs might be a determinant for A-EJ choice. To facilitate efficient repair of high-complexity DSBs, A-EJ employs distinct molecular patterns such as longer micro-homologous junctions and non-templated nucleotide addition. Furthermore, the A-EJ choice is modulated by the degree of homology near DSB loci, competing with homologous recombination machinery. These findings further enhance the understanding of A-EJ/alt-NHEJ pathway choice.

DNA double-strand break repair by homologous recombination has a specialised role in meiosis by generating crossovers that enable the formation of haploid germ cells. This requires meiosis-specific MEILB2-BRME1, which interacts with BRCA2 to facilitate loading of recombinases onto resected DNA ends. Here, we report the crystal structure of the MEILB2-BRME1 2:2 core complex, revealing a parallel four-helical assembly that recruits BRME1 to meiotic double-strand breaks in vivo. It forms an N-terminal β-cap that binds to DNA, and a MEILB2 coiled-coil that bridges to C-terminal ARM domains. Upon BRCA2-binding, MEILB2-BRME1 2:2 complexes dimerize into a V-shaped 2:4:4 complex, with rod-like MEILB2-BRME1 components arranged at right-angles. The β-caps located at the tips of the MEILB2-BRME1 limbs are separated by 25 nm, allowing them to bridge between DNA molecules. Thus, we propose that BRCA2 induces MEILB2-BRME1 to function as a DNA clamp, connecting resected DNA ends or homologous chromosomes to facilitate meiotic recombination.

Phase separation forms membraneless compartments in the nuclei, including by establishing heterochromatin \"domains\" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination, and \"safe\" homologous recombination (HR) repair of these sequences requires the movement of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation to these dynamics is unclear. Here, we show that Nup98 nucleoporin is recruited to heterochromatic repair sites before relocalization through Sec13 or Nup88 nucleoporins, and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing a novel \"off-pore\" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote.

The model haloarchaeon Haloferax volcanii is polyploid with about 20 copies of its major chromosome. Recently it has been described that highly efficient intermolecular gene conversion operates in H. volcanii to equalize the chromosomal copies. In the current study, 24 genes were selected that encode proteins with orthologs involved in gene conversion or homologous recombination in archaea, bacteria, or eukaryotes. Single gene deletion strains of 22 genes and a control gene were constructed in two parent strains for a gene conversion assay; only radA and radB were shown to be essential. Protoplast fusions were used to generate strains that were heterozygous for the gene HVO_2528, encoding an enzyme for carotinoid biosynthesis. It was revealed that a lack of six of the proteins did not influence the efficiency of gene conversion, while sixteen mutants had severe gene conversion defects. Notably, lack of paralogous proteins of gene families had very different effects, e.g., mutant Δrad25b had no phenotype, while mutants Δrad25a, Δrad25c, and Δrad25d were highly compromised. Generation of a quadruple rad25 and a triple sph deletion strain also indicated that the paralogs have different functions, in contrast to sph2 and sph4, which cannot be deleted simultaneously. There was no correlation between the severity of the phenotypes and the respective transcript levels under non-stressed conditions, indicating that gene expression has to be induced at the onset of gene conversion. Phylogenetic trees of the protein families Rad3/25, MutL/S, and Sph/SMC/Rad50 were generated to unravel the history of the paralogous proteins of H. volcanii. Taken together, unselected intermolecular gene conversion in H. volcanii involves at least 16 different proteins, the molecular roles of which can be studied in detail in future projects.

BACKGROUND: The obligate intracellular bacterial family Chlamydiaceae comprises a number of different species that cause disease in various vertebrate hosts including humans. Chlamydia suis, primarily found in the gastrointestinal tract of pigs, is the only species of the Chlamydiaceae family to have naturally gained tetracycline resistance (TetR), through a genomic island (Tet-island), integrated into the middle of chromosomal invasin-like gene inv. Previous studies have hypothesised that the uptake of the Tet-island from a host outside the Chlamydiaceae family was a unique event, followed by spread among C. suis through homologous recombination. In vitro recombination studies have shown that Tet-island exchange between C. suis strains is possible. Our aim in this study was to gain a deeper understanding of the interclade recombination of the Tet-island, among currently circulating C. suis field strains compared to in vitro-generated recombinants, using published whole genome sequences of C. suis field strains (n = 35) and in vitro-generated recombinants (n = 63).
RESULTS: We found that the phylogeny of inv better reflected the phylogeny of the Tet-island than that of the whole genome, supporting recombination rather than site-specific insertion as the means of transfer. There were considerable differences between the distribution of recombinations within in vitro-generated strains compared to that within the field strains. These differences are likely because in vitro-generated recombinants were selected for a tetracycline and rifamycin/rifampicin resistant background, leading to the largest peak of recombination across the Tet-island. Finally, we found that interclade recombinations across the Tet-island were more variable in length downstream of the Tet-island than upstream.
CONCLUSIONS: Our study supports the hypothesis that the occurrence of TetR strains in both clades of C. suis came about through interclade recombination after a single ancestral horizontal gene transfer event.

OBJECTIVE: Our study aimed to develop and validate a homologous recombination deficiency (HRD) scoring algorithm in the Chinese breast cancer population.
METHODS: Ninety-six in-house breast cancer (BC) samples and 6 HRD-positive standard cells were analyzed by whole-genome sequencing (WGS). Besides, 122 BCs from the TCGA database were down-sampled to ~ 1X WGS. We constructed an algorithm named AcornHRD for HRD score calculated based on WGS at low coverage as input data to estimate large-scale copy number alteration (LCNA) events on the genome. A clinical cohort of 50 BCs (15 cases carrying BRCA mutation) was used to assess the association between HRD status and anthracyclines-based neoadjuvant treatment outcomes.
RESULTS: A 100-kb window was defined as the optimal size using 41 in-house cases and the TCGA dataset. HRD score high threshold was determined as HRD score ≥ 10 using 55 in-house BCs with BRCA mutation to achieve a 95% BRCA-positive agreement rate. Furthermore, the HRD status agreement rate of AcornHRD is 100%, while the ShallowHRD is 60% in standard cells. BRCA mutation was significantly associated with a high HRD score evaluated by AcornHRD and ShallowHRD (p = 0.008 and p = 0.003, respectively) in the TCGA dataset. However, AcornHRD showed a higher positive agreement rate than did the ShallowHRD algorithm (70% vs 60%). In addition, the BRCA-positive agreement rate of AcornHRD was superior to that of ShallowHRD (87% vs 13%) in the clinical cohort. Importantly, the high HRD score assessed by AcornHRD was significantly correlated with a residual cancer burden score of 0 or 1 (RCB0/1). Besides, the HRD-positive group was more likely to respond to anthracycline-based chemotherapy than the HRD-negative group (pCR [OR = 9.5, 95% CI 1.11-81.5, p = 0.040] and RCB0/1 [OR = 10.29, 95% CI 2.02-52.36, p = 0.005]).
CONCLUSIONS: Using the AcornHRD algorithm evaluation, our analysis demonstrated the high performance of the LCNA genomic signature for HRD detection in breast cancers.

BACKGROUND: Homologous recombination deficiency (HRD) stands as a clinical indicator for discerning responsive outcomes to platinum-based chemotherapy and poly ADP-ribose polymerase (PARP) inhibitors. One of the conventional approaches to HRD prognostication has generally centered on identifying deleterious mutations within the BRCA1/2 genes, along with quantifying the genomic scars, such as Genomic Instability Score (GIS) estimation with scarHRD. However, the scarHRD method has limitations in scenarios involving tumors bereft of corresponding germline data. Although several RNA-seq-based HRD prediction algorithms have been developed, they mainly support cohort-wise classification, thereby yielding HRD status without furnishing an analogous quantitative metric akin to scarHRD. This study introduces the expHRD method, which operates as a novel transcriptome-based framework tailored to n-of-1-style HRD scoring.
RESULTS: The prediction model has been established using the elastic net regression method in the Cancer Genome Atlas (TCGA) pan-cancer training set. The bootstrap technique derived the HRD geneset for applying the expHRD calculation. The expHRD demonstrated a notable correlation with scarHRD and superior performance in predicting HRD-high samples. We also performed intra- and extra-cohort evaluations for clinical feasibility in the TCGA-OV and the Genomic Data Commons (GDC) ovarian cancer cohort, respectively. The innovative web service designed for ease of use is poised to extend the realms of HRD prediction across diverse malignancies, with ovarian cancer standing as an emblematic example.
CONCLUSIONS: Our novel approach leverages the transcriptome data, enabling the prediction of HRD status with remarkable precision. This innovative method addresses the challenges associated with limited available data, opening new avenues for utilizing transcriptomics to inform clinical decisions.

Although more difficult to detect than in the cytoplasm, it is now clear that actin polymerization occurs in the nucleus and that it plays a role in the specific processes of the nucleus such as transcription, replication, and DNA repair. A number of studies suggest that nuclear actin polymerization is promoting precise DNA repair by homologous recombination, which could potentially be of help for precise genome editing and gene therapy. This review summarizes the findings and describes the challenges and chances in the field.