The histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSα.

DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 trimethyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.

Mutant huntingtin protein induces MLH1 degradation, DNA hyperexcision, and cGAS-STING-dependent apoptosis.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The repeat-expanded HTT encodes a mutated HTT (mHTT), which is known to induce DNA double-strand breaks (DSBs), activation of the cGAS-STING pathway, and apoptosis in HD. However, the mechanism by which mHTT triggers these events is unknown. Here, we show that HTT interacts with both exonuclease 1 (Exo1) and MutLα (MLH1-PMS2), a negative regulator of Exo1. While the HTT-Exo1 interaction suppresses the Exo1-catalyzed DNA end resection during DSB repair, the HTT-MutLα interaction functions to stabilize MLH1. However, mHTT displays a significantly reduced interaction with Exo1 or MutLα, thereby losing the ability to regulate Exo1. Thus, cells expressing mHTT exhibit rapid MLH1 degradation and hyperactive DNA excision, which causes severe DNA damage and cytosolic DNA accumulation. This activates the cGAS-STING pathway to mediate apoptosis. Therefore, we have identified unique functions for both HTT and mHTT in modulating DNA repair and the cGAS-STING pathway-mediated apoptosis by interacting with MLH1. Our work elucidates the mechanism by which mHTT causes HD.

The mismatch recognition protein MutSα promotes nascent strand degradation at stalled replication forks.

Mismatch repair (MMR) is a replication-coupled DNA repair mechanism and plays multiple roles at the replication fork. The well-established MMR functions include correcting misincorporated nucleotides that have escaped the proofreading activity of DNA polymerases, recognizing nonmismatched DNA adducts, and triggering a DNA damage response. In an attempt to determine whether MMR regulates replication progression in cells expressing an ultramutable DNA polymerase ɛ (Polɛ), carrying a proline-to-arginine substitution at amino acid 286 (Polɛ-P286R), we identified an unusual MMR function in response to hydroxyurea (HU)-induced replication stress. Polɛ-P286R cells treated with hydroxyurea exhibit increased MRE11-catalyzed nascent strand degradation. This degradation by MRE11 depends on the mismatch recognition protein MutSα and its binding to stalled replication forks. Increased MutSα binding at replication forks is also associated with decreased loading of replication fork protection factors FANCD2 and BRCA1, suggesting blockage of these fork protection factors from loading to replication forks by MutSα. We find that the MutSα-dependent MRE11-catalyzed fork degradation induces DNA breaks and various chromosome abnormalities. Therefore, unlike the well-known MMR functions of ensuring replication fidelity, the newly identified MMR activity of promoting genome instability may also play a role in cancer avoidance by eliminating rogue cells.

Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells.

Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, and chromatin immunoprecipitation sequencing analyses to determine their response to the treatment. We show that the mismatch repair processing of oxidative DNA damage involves cohesive interactions between mismatch recognition protein MutSα, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSα or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSα and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSα to ATM. Consistently, MutSα and SETD2 are highly coenriched at oxidative damage sites. The data presented here support a model wherein MutSα, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage.

DNA polymerase θ promotes CAG•CTG repeat expansions in Huntington's disease via insertion sequences of its catalytic domain.

Huntington's disease (HD), a neurodegenerative disease characterized by progressive dementia, psychiatric problems, and chorea, is known to be caused by CAG repeat expansions in the HD gene HTT. However, the mechanism of this pathology is not fully understood. The translesion DNA polymerase θ (Polθ) carries a large insertion sequence in its catalytic domain, which has been shown to allow DNA loop-outs in the primer strand. As a result of high levels of oxidative DNA damage in neural cells and Polθ's subsequent involvement in base excision repair of oxidative DNA damage, we hypothesized that Polθ contributes to CAG repeat expansion while repairing oxidative damage within HTT. Here, we performed Polθ-catalyzed in vitro DNA synthesis using various CAG•CTG repeat DNA substrates that are similar to base excision repair intermediates. We show that Polθ efficiently extends (CAG)n•(CTG)n hairpin primers, resulting in hairpin retention and repeat expansion. Polθ also triggers repeat expansions to pass the threshold for HD when the DNA template contains 35 repeats upward. Strikingly, Polθ depleted of the catalytic insertion fails to induce repeat expansions regardless of primers and templates used, indicating that the insertion sequence is responsible for Polθ's error-causing activity. In addition, the level of chromatin-bound Polθ in HD cells is significantly higher than in non-HD cells and exactly correlates with the degree of CAG repeat expansion, implying Polθ's involvement in triplet repeat instability. Therefore, we have identified Polθ as a potent factor that promotes CAG•CTG repeat expansions in HD and other neurodegenerative disorders.

OTUB1 stabilizes mismatch repair protein MSH2 by blocking ubiquitination.

DNA mismatch repair (MMR) maintains genome stability primarily by correcting replication errors. MMR deficiency can lead to cancer development and bolsters cancer cell resistance to chemotherapy. However, recent studies have shown that checkpoint blockade therapy is effective in MMR-deficient cancers, thus the ability to identify cancer etiology would greatly benefit cancer treatment. MutS homolog 2 (MSH2) is an obligate subunit of mismatch recognition proteins MutSα (MSH2-MSH6) and MutSß (MSH2-MSH3). Precise regulation of MSH2 is critical, as either over- or underexpression of MSH2 results in an increased mutation frequency. The mechanism by which cells maintain MSH2 proteostasis is unknown. Using functional ubiquitination and deubiquitination assays, we show that the ovarian tumor (OTU) family deubiquitinase ubiquitin aldehyde binding 1 (OTUB1) inhibits MSH2 ubiquitination by blocking the E2 ligase ubiquitin transfer activity. Depleting OTUB1 in cells promotes the ubiquitination and subsequent degradation of MSH2, leading to greater mutation frequency and cellular resistance to genotoxic agents, including the common chemotherapy agents N-methyl-N'-nitro-N-nitrosoguanidine and cisplatin. Taken together, our data identify OTUB1 as an important regulator of MSH2 stability and provide evidence that OTUB1 is a potential biomarker for cancer etiology and therapy.

HDAC6 deacetylates and ubiquitinates MSH2 to maintain proper levels of MutSα.

MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSß) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.

NaCl induces flocculation and lipid oxidation of soybean oil body emulsions recovered by neutral aqueous extraction.

BACKGROUND: Soybean oil bodies (SOB) are naturally pre-emulsified lipid droplets recovered directly from soybean seeds. Almost all food emulsions contain salts. However, it was not clear how the incorporation of salts affected the physicochemical stability of SOB. RESULTS: This study investigated the effect of NaCl (0-1.2%) on the physical and oxidative stability of SOB emulsions under neutral (pH 7) and acidic (pH 3) conditions. In the presence of NaCl, the SOB emulsion (pH 7) showed strong flocculation during storage due to electrostatic screening. The NaCl-induced flocculation of SOB was attenuated at pH 3, which may be due to the difference in conformation or interaction of the protein interfaces covering SOB at different pH values. The increase in ionic strength or acid conditioning treatment resulted in a remarkable increase in the stability of SOB emulsions against coalescence. The confocal laser scanning microscopy images also confirmed the NaCl-induced changes in the flocculation/coalescence properties of SOB. The oxidative behavior tests indicated that SOB emulsions containing NaCl were more susceptible to lipid oxidation but protein oxidation was inhibited due to electrostatic screening, which reduced pro-oxidant accessibility of unadsorbed proteins in the emulsion. This oxidative behavior was attenuated at pH 3. CONCLUSION: The incorporation of NaCl significantly reduced the physical and oxidative stability of the SOB emulsion, and acidic pH mitigated NaCl-induced flocculation and lipid oxidation of SOB. © 2021 Society of Chemical Industry.

Cancer-driving H3G34V/R/D mutations block H3K36 methylation and H3K36me3-MutSα interaction.

Somatic mutations on glycine 34 of histone H3 (H3G34) cause pediatric cancers, but the underlying oncogenic mechanism remains unknown. We demonstrate that substituting H3G34 with arginine, valine, or aspartate (H3G34R/V/D), which converts the non-side chain glycine to a large side chain-containing residue, blocks H3 lysine 36 (H3K36) dimethylation and trimethylation by histone methyltransferases, including SETD2, an H3K36-specific trimethyltransferase. Our structural analysis reveals that the H3 "G33-G34" motif is recognized by a narrow substrate channel, and that H3G34/R/V/D mutations impair the catalytic activity of SETD2 due to steric clashes that impede optimal SETD2-H3K36 interaction. H3G34R/V/D mutations also block H3K36me3 from interacting with mismatch repair (MMR) protein MutSα, preventing the recruitment of the MMR machinery to chromatin. Cells harboring H3G34R/V/D mutations display a mutator phenotype similar to that observed in MMR-defective cells. Therefore, H3G34R/V/D mutations promote genome instability and tumorigenesis by inhibiting MMR activity.

Phosphorylation of proliferating cell nuclear antigen promotes cancer progression by activating the ATM/Akt/GSK3ß/Snail signaling pathway.

Proliferating cell nuclear antigen (PCNA) and its posttranslational modifications regulate DNA metabolic reactions, including DNA replication and repair, at replication forks. PCNA phosphorylation at Tyr-211 (PCNA-Y211p) inhibits DNA mismatch repair and induces misincorporation during DNA synthesis. Here, we describe an unexpected role of PCNA-Y211p in cancer promotion and development. Cells expressing phosphorylation-mimicking PCNA, PCNA-Y211D, show elevated hallmarks specific to the epithelial-mesenchymal transition (EMT), including the up-regulation of the EMT-promoting factor Snail and the down-regulation of EMT-inhibitory factors E-cadherin and GSK3ß. The PCNA-Y211D-expressing cells also exhibited active cell migration and underwent G2/M arrest. Interestingly, all of these EMT-associated activities required the activation of ATM and Akt kinases, as inactivating these protein kinases by gene knockdown or inhibitors blocked EMT-associated signaling and cell migration. We concluded that PCNA phosphorylation promotes cancer progression via the ATM/Akt/GSK3ß/Snail signaling pathway. In conclusion, this study identifies a novel PCNA function and reveals the molecular basis of phosphorylated PCNA-mediated cancer development and progression.

H3K36me3-mediated mismatch repair preferentially protects actively transcribed genes from mutation.

Histone H3 trimethylation at lysine 36 (H3K36me3) is an important histone mark involved in both transcription elongation and DNA mismatch repair (MMR). It is known that H3K36me3 recruits the mismatch-recognition protein MutSα to replicating chromatin via its physical interaction with MutSα's PWWP domain, but the exact role of H3K36me3 in transcription is undefined. Using ChIP combined with whole-genome DNA sequencing analysis, we demonstrate here that H3K36me3, together with MutSα, is involved in protecting against mutation, preferentially in actively transcribed genomic regions. We found that H3K36me3 and MutSα are much more co-enriched in exons and actively transcribed regions than in introns and nontranscribed regions. The H3K36me3-MutSα co-enrichment correlated with a much lower mutation frequency in exons and actively transcribed regions than in introns and nontranscribed regions. Correspondingly, depleting H3K36me3 or disrupting the H3K36me3-MutSα interaction elevated the spontaneous mutation frequency in actively transcribed genes, but it had little influence on the mutation frequency in nontranscribed or transcriptionally inactive regions. Similarly, H2O2-induced mutations, which mainly cause base oxidations, preferentially occurred in actively transcribed genes in MMR-deficient cells. The data presented here suggest that H3K36me3-mediated MMR preferentially safeguards actively transcribed genes not only during replication by efficiently correcting mispairs in early replicating chromatin but also during transcription by directly or indirectly removing DNA lesions associated with a persistently open chromatin structure.

[UPLC fingerprint and multi-components content determination of different processed products of Angelica sinensis].

Ten batches of Angelica sinensis from three producing areas( Tuoxiang,Minxian and Weiyuan of Gansu province) were selected as the research objects,and processed into raw A. sinensis,A. sinensis with alcohol,and A. sinensis with soil respectively through the standard processing methods. Ultra-high performance liquid chromatography( UPLC) was used to establish fingerprint for three processed products of A. sinensis,and determine the contents of 9 phenolic acids and phthalide compounds. The similarity was analyzed with Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine,which showed that the chromatographic peaks of the same processed samples of A. sinensis were basically similar,with all similarities greater than 0. 950. The difference between different processed products and their control spectra was not obvious,with all similarities also higher than 0. 950.On the basis of using principal component analysis( PCA) and OPLS-DA to seek the difference components between groups,the improved distance coefficient method can be used to effectively distinguish the three processed products of A. sinensis by fingerprint similarity. At the same time,the determination method of nine phenolic acids and phthalide in A. sinensis was established by UPLC,and the comparison between different processed products was carried out. The results showed that the content of various components was changed as compared with the raw A. sinensis. The contents of coniferyl ferulate and ligustilide in the A. sinensis with alcohol were increased significantly,and the content of coniferyl ferulate was obviously increased in A. sinensis with soil. The method established in this paper can effectively distinguish different processed products of A. sinensis and determine the content of the main components in them.

Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis.

Proliferating cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSß and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function.

Mechanism of Curcuma wenyujin Rhizoma on Acute Blood Stasis in Rats Based on a UPLC-Q/TOF-MS Metabolomics and Network Approach.

Rhizome of Curcuma wenyujin, which is called EZhu in China, is a traditional Chinese medicine used to treat blood stasis for many years. However, the underlying mechanism of EZhu is not clear at present. In this study, plasma metabolomics combined with network pharmacology were used to elucidate the therapeutic mechanism of EZhu in blood stasis from a metabolic perspective. The results showed that 26 potential metabolite markers of acute blood stasis were screened, and the levels were all reversed to different degrees by EZhu preadministration. Metabolic pathway analysis showed that the improvement of blood stasis by Curcuma wenyujin rhizome was mainly related to lipid metabolism (linoleic acid metabolism, ether lipid metabolism, sphingolipid metabolism, glycerophospholipid metabolism, and arachidonic acid metabolism) and amino acid metabolisms (tryptophan metabolism, lysine degradation). The component-target-pathway network showed that 68 target proteins were associated with 21 chemical components in EZhu. Five metabolic pathways of the network, including linoleic acid metabolism, sphingolipid metabolism, glycerolipid metabolism, arachidonic acid metabolism, and steroid hormone biosynthesis, were consistent with plasma metabolomics results. In conclusion, plasma metabolomics combined with network pharmacology can be helpful to clarify the mechanism of EZhu in improving blood stasis and to provide a literature basis for further research on the therapeutic mechanism of EZhu in clinical practice.

[UPLC fingerprint and multi-components determination of three processed products of Rhizome of Curcuma wenyujin].

To analyse the quality of three processed products of Rhizome of Curcuma wenyujin by establishing an ultra performance liquid chromatography (UPLC) method for simultaneously determining five sesquiterpene components in three processed products of rhizome of C. wenyujin and establishing UPLC fingerprints. Component determination was achieved on Waters ACQUITY UPLC BEH C18 column（2.1 mm×50 mm, 1.7 µm), with acetonitrile-water as mobile phase for gradient elution. The flow rate was 0.3 mL·min⁻¹; column temperature was 30 °C; the detection wavelength was set at 214 nm and injection volume was 1 µL. The similarity was analyzed with "Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica (2012.130723)", and hydrophobic cluster analysis (HCA), principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted by using simca-p14.1 software to investigate the differences in components among these three kinds of processed products. The curzerene, curdione, curcumol, germacrone, furanodiene and ß-elemene showed good linearity relationship with chromatographic peak area within the ranges of 10.8-320（r=0.999 9）, 10.36-259（r=0.998 1）, 10.54-263.5（r=0.999 3）, 30.2-755（r=0.999 6）and 34.38-862（r=0.999 9）mg·L⁻¹, respectively; their average recoveries were 98.75%, 98.69%, 98.63%, 99.76% and 99.57% respectively, with RSD of 2.67%, 1.47%, 1.29%, 2.54% and 0.87% respectively. The similarity of 30 batches of samples was larger than 0.9, indicating good consistency of the samples. The samples can be clearly classified into three categories for HCA, PCA and OPLS-DA pattern recognition, the differential chromatographic peak among three processed products was found respectively. The results showed that the pharmacology basis had changed obviously after processing of Rhizome of C. wenyujin, so it can provide the scientific basis for rational clinical application and establishing quality standards of three processed products of Rhizome of curcuma wenyujin.

Arsenic Inhibits DNA Mismatch Repair by Promoting EGFR Expression and PCNA Phosphorylation.

Both genotoxic and non-genotoxic chemicals can act as carcinogens. However, while genotoxic compounds lead directly to mutations that promote unregulated cell growth, the mechanism by which non-genotoxic carcinogens lead to cellular transformation is poorly understood. Using a model non-genotoxic carcinogen, arsenic, we show here that exposure to arsenic inhibits mismatch repair (MMR) in human cells, possibly through its ability to stimulate epidermal growth factor receptor (EGFR)-dependent tyrosine phosphorylation of proliferating cellular nuclear antigen (PCNA). HeLa cells exposed to exogenous arsenic demonstrate a dose- and time-dependent increase in the levels of EGFR and tyrosine 211-phosphorylated PCNA. Cell extracts derived from arsenic-treated HeLa cells are defective in MMR, and unphosphorylated recombinant PCNA restores normal MMR activity to these extracts. These results suggest a model in which arsenic induces expression of EGFR, which in turn phosphorylates PCNA, and phosphorylated PCNA then inhibits MMR, leading to increased susceptibility to carcinogenesis. This study suggests a putative novel mechanism of action for arsenic and other non-genotoxic carcinogens.

Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-stranded RNA.

3' repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T. Kinetic measurements indicate that the apparent Km of TREX1 for RNA is higher than that for DNA. Like RNase T, human TREX1 is active in degrading native tRNA substrates. Previously reported TREX1 crystal structures have revealed that the substrate binding sites are open enough to accommodate the extra hydroxyl group in RNA, further supporting our conclusion that TREX1 acts on RNA. These findings indicate that its RNase activity needs to be taken into account when evaluating the physiological role of TREX1.

Coordinated processing of 3' slipped (CAG)n/(CTG)n hairpins by DNA polymerases ß and δ preferentially induces repeat expansions.

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3'-slipped hairpin using its 3'-5' proofreading activity when the hairpin contains no immediate 3' complementary sequences. However, in the presence of pol ß, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol ß incorporates several nucleotides to the hairpin 3'-end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3'-slipped (CAG)n/(CTG)n hairpins by polymerases δ and ß on during DNA synthesis induces CAG/CTG repeat expansions.

Bibliometric analysis of kinship analysis from 1960 to 2023: global trends and development.

Kinship analysis is a crucial aspect of forensic genetics. This study analyzed 1,222 publications on kinship analysis from 1960 to 2023 using bibliometric analysis techniques, investigating the annual publication and citation patterns, most productive countries, organizations, authors and journals, most cited documents and co-occurrence of keywords. The initial publication in this field occurred in 1960. Since 2007, there has been a significant increase in publications, with over 30 published annually except for 2010. China had the most publications (n = 213, 17.43%), followed by the United States (n = 175, 14.32%) and Germany (n = 89, 7.28%). The United States also had the highest citation count. Sichuan University in China has the largest number of published articles. The University of Leipzig and the University of Cologne in Germany exhibit the highest total citation count and average citation, respectively. Budowle B was the most prolific author and Kayser M was the most cited author. In terms of publications, Forensic Science International- Genetics, Forensic Science International, and International Journal of Legal Medicine were the most prolific journals. Among them, Forensic Science International-Genetics boasted the highest h-index, citation count, and average citation rate. The most frequently cited publication was "Van Oven M, 2009, Hum Mutat", with a total of 1,361 citations. The most frequent co-occurrence keyword included "DNA", "Loci", "Paternity testing", "Population", "Markers", and "Identification", with recent interest focusing on "Kinship analysis", "SNP" and "Inference". The current research is centered around microhaplotypes, forensic genetic genealogy, and massively parallel sequencing. The field advanced with new DNA analysis methods, tools, and genetic markers. Collaborative research among nations, organizations, and authors benefits idea exchange, problem-solving efficiency, and high-quality results.