New insights into ATR inhibition in muscle invasive bladder cancer: The role of apolipoprotein B mRNA editing catalytic subunit 3B.

Background: Apolipoprotein B mRNA editing catalytic polypeptide (APOBEC), an endogenous mutator, induces DNA damage and activates the ataxia telangiectasia and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) pathway. Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer (MIBC), it has a poor survival rate. Therefore, this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B (APOBEC3B) expressing MIBC. Methods: Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC. The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis. Western blot analysis was performed to confirm differences in phosphorylated Chk1 (pChk1) expression according to the APOBEC3B expression. Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin. Conclusion: There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC. Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels. Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression. Compared to cisplatin single treatment, combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression. Conclusion: Our study shows that APOBEC3B's higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition. This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.

Papillary Thyroid Cancer Remodels the Genetic Information Processing Pathways.

The genetic causes of the differentiated, highly treatable, and mostly non-fatal papillary thyroid cancer (PTC) are not yet fully understood. The mostly accepted PTC etiology blames the altered sequence or/and expression level of certain biomarker genes. However, tumor heterogeneity and the patient's unique set of favoring factors question the fit-for-all gene biomarkers. Publicly accessible gene expression profiles of the cancer nodule and the surrounding normal tissue from a surgically removed PTC tumor were re-analyzed to determine the cancer-induced alterations of the genomic fabrics responsible for major functional pathways. Tumor data were compared with those of standard papillary and anaplastic thyroid cancer cell lines. We found that PTC regulated numerous genes associated with DNA replication, repair, and transcription. Results further indicated that changes of the gene networking in functional pathways and the homeostatic control of transcript abundances also had major contributions to the PTC phenotype occurrence. The purpose to proliferate and invade the entire gland may explain the substantial transcriptomic differences we detected between the cells of the cancer nodule and those spread in homo-cellular cultures (where they need only to survive). In conclusion, the PTC etiology should include the complex molecular mechanisms involved in the remodeling of the genetic information processing pathways.

Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.

Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.

Genome-wide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression.

BACKGROUND: DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know how transcription influences overall DNA replication progression. RESULTS: To characterize sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase to identify replication fork/stalling hotspots as replication progresses through the genome. These sites are typically associated with increased DNA damage, overlapped with fragile sites and with breakpoints of rearrangements identified in cancers but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. Indeed, we find that slowing down transcription elongation slows down directly replication progression through genes. This indicates that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. At the same time, we find instances where transcription activity favors replication progression because it reduces histone density. CONCLUSIONS: Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.

Reduced Levels of Lagging Strand Polymerases Shape Stem Cell Chromatin.

Stem cells display asymmetric histone inheritance while non-stem progenitor cells exhibit symmetric patterns in the Drosophila male germline lineage. Here, we report that components involved in lagging strand synthesis, such as DNA polymerase α and δ (Polα and Polδ), have significantly reduced levels in stem cells compared to progenitor cells. Compromising Polα genetically induces the replication-coupled histone incorporation pattern in progenitor cells to be indistinguishable from that in stem cells, which can be recapitulated using a Polα inhibitor in a concentration-dependent manner. Furthermore, stem cell-derived chromatin fibers display a higher degree of old histone recycling by the leading strand compared to progenitor cell-derived chromatin fibers. However, upon reducing Polα levels in progenitor cells, the chromatin fibers now display asymmetric old histone recycling just like GSC-derived fibers. The old versus new histone asymmetry is comparable between stem cells and progenitor cells at both S-phase and M-phase. Together, these results indicate that developmentally programmed expression of key DNA replication components is important to shape stem cell chromatin. Furthermore, manipulating one crucial DNA replication component can induce replication-coupled histone dynamics in non-stem cells in a manner similar to that in stem cells.

CRISPR-Cas13d screens identify KILR, a breast cancer risk-associated lncRNA that regulates DNA replication and repair.

BACKGROUND: Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered 844 lncRNAs that were genetically linked to breast cancer through genome-wide association studies (GWAS). Here, we show that a subset of these lncRNAs alter breast cancer risk by modulating cell proliferation, and provide evidence that a reduced expression on one lncRNA increases breast cancer risk through aberrant DNA replication and repair. METHODS: We performed pooled CRISPR-Cas13d-based knockdown screens in breast cells to identify which of the 844 breast cancer-associated lncRNAs alter cell proliferation. We selected one of the lncRNAs that increased cell proliferation, KILR, for follow-up functional studies. KILR pull-down followed by mass spectrometry was used to identify binding proteins. Knockdown and overexpression studies were performed to assess the mechanism by which KILR regulates proliferation. RESULTS: We show that KILR functions as a tumor suppressor, safeguarding breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. Mechanistically, KILR sequesters RPA1, a subunit of the RPA complex required for DNA replication and repair. Reduced KILR expression promotes breast cancer cell proliferation by increasing the available pool of RPA1 and speed of DNA replication. Conversely, KILR overexpression promotes apoptosis in breast cancer cells, but not normal breast cells. CONCLUSIONS: Our results confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

Beyond membrane permeability: A role for the small RNA MicF in regulation of chromosome replication and partitioning.

Small regulatory RNAs (sRNA) have been shown to play a large role in the management of stress responses in Escherichia coli and other bacteria. sRNAs act post-transcriptionally on target mRNA through an imperfect base pairing mechanism to regulate downstream protein expression. The imperfect base pairing allows a single sRNA to bind and regulate a variety mRNA targets which can form intricate regulatory networks that connect different physiological processes for the cell's response. Upon exposure to antimicrobials and superoxide generating agents, the MicF sRNA in E. coli has been shown to regulate a small set of genes involved in the management of membrane permeability. Currently, it is unknown whether MicF acts on other processes to mediate the response to these agents. Using an sRNA interaction prediction tool, we identified genes in E. coli that are potentially regulated by MicF. Through subsequent analysis using a sfGFP-based reporter-gene fusion, we have validated two novel targets of MicF regulation: SeqA, a negative modulator of DNA replication, and ObgE, a GTPase crucial for chromosome partitioning. Importantly, the interaction between MicF and these target mRNAs is contingent upon the presence of the RNA chaperone protein, Hfq. Furthermore, our findings affirm the role of MicF's conserved 5' seed pairing region in initiating these regulatory interactions. Our study suggests that, beyond its established role in membrane permeability management, MicF exerts control over chromosome dynamics in response to distinct environmental cues, implicating a more multifaceted regulatory function in bacterial stress adaptation.

Molecular insights into the prototypical single-stranded DNA-binding protein from E. coli.

The SSB protein of Escherichia coli functions to bind single-stranded DNA wherever it occurs during DNA metabolism. Depending upon conditions, SSB occurs in several different binding modes. In the course of its function, SSB diffuses on ssDNA and transfers rapidly between different segments of ssDNA. SSB interacts with many other proteins involved in DNA metabolism, with 22 such SSB-interacting proteins, or SIPs, defined to date. These interactions chiefly involve the disordered and conserved C-terminal residues of SSB. When not bound to ssDNA, SSB can aggregate to form a phase-separated biomolecular condensate. Current understanding of the properties of SSB and the functional significance of its many intermolecular interactions are summarized in this review.

Investigation into in silico and in vitro approaches for inhibitors targeting MCM10 in Leishmania donovani: a comprehensive study.

Visceral Leishmaniasis (VL), the second neglected tropical disease caused by various Leishmania species, presents a significant public health challenge due to limited treatment options and the absence of vaccines. The agent responsible for visceral leishmaniasis, also referred to as "black fever" in India, is Leishmania donovani. This study focuses on L. donovani Minichromosome maintenance 10 (LdMcm10), a crucial protein in the DNA replication machinery, as a potential therapeutic target in Leishmania therapy using in silico and in vitro approaches. We employed bioinformatics tools, molecular docking, and molecular dynamics simulations to predict potential inhibitors against the target protein. The research revealed that the target protein lacks homologues in the host, emphasizing its potential as a drug target. Ligands from the DrugBank database were screened against LdMcm10 using PyRx software. The top three compounds, namely suramin, vapreotide, and pasireotide, exhibiting the best docking scores, underwent further investigation through molecular dynamic simulation and in vitro analysis. The observed structural dynamics suggested that LdMcm10-ligand complexes maintained consistent binding throughout the 300 ns simulation period, with minimal variations in their backbone. These findings suggest that these three compounds hold promise as potential lead compounds for developing new drugs against leishmaniasis. In vitro experiments also demonstrated a dose-dependent reduction in L. donovani viability for suramin, vapreotide, and pasireotide, with computed IC50 values providing quantitative metrics of their anti-leishmanial efficacy. The research offers a comprehensive understanding of LdMcm10 as a drug target and provides a foundation for further investigations and clinical exploration, ultimately advancing drug discovery strategies for leishmaniasis treatment.

Functional studies in yeast confirm the pathogenicity of a new GINS3 Meier-Gorlin syndrome variant.

Meier-Gorlin syndrome (MGORS) is an autosomal recessive disorder characterized by short stature, microtia, and patellar hypoplasia, and is caused by pathogenic variants of cellular factors involved in the initiation of DNA replication. We previously reported that biallelic variants in GINS3 leading to amino acid changes at position 24 (p.Asp24) cause MGORS. Here, we describe the phenotype of a new individual homozygous for the Asp24Asn variant. We also report the clinical characteristics of an individual harboring a novel homozygous GINS3 variant (Ile25Phe) and features suggestive of MGORS. Modification of the corresponding residue in yeast Psf3 (Val9Phe) compromised S phase progression compared to a humanized Psf3 Val9Ile variant. Expression of Psf3 Val9Phe in yeast also caused sensitivity to elevated temperature and the replicative stress-inducing drug hydroxyurea, confirming partial loss of function of this variant in vivo and allowing us to upgrade the classification of this variant. Taken together, these data validate the critical importance of the GINS DNA replication complex in the molecular etiology of MGORS.

H2B oncohistones cause homologous recombination defect and genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe.

Canonical oncohistones are histone H3 mutations in the N-terminal tail associated with tumors and affect gene expression by altering H3 post-translational modifications (PTMs) and the epigenetic landscape. Noncanonical oncohistone mutations occur in both tails and globular domains of all four core histones and alter gene expression by perturbing chromatin remodeling. However, the effects and mechanisms of noncanonical oncohistones remain largely unknown. Here we characterized 16 noncanonical H2B oncohistones in the fission yeast Schizosaccharomyces pombe. We found that seven of them exhibited temperature sensitivities and 11 exhibited genotoxic sensitivities. A detailed study of two of these onco-mutants H2BG52D and H2BP102L revealed that they were defective in homologous recombination (HR) repair with compromised histone eviction and Rad51 recruitment. Interestingly, their genotoxic sensitivities and HR defects were rescued by the inactivation of the H2BK119 deubiquitination function of Ubp8 in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. The levels of H2BK119 monoubiquitination (H2Bub) in the H2BG52D and H2BP102L mutants are reduced in global genome and at local DNA break sites presumably due to enhanced recruitment of Ubp8 onto nucleosomes and are recovered upon loss of H2B deubiquitination function of the SAGA complex. Moreover, H2BG52D and H2BP102L heterozygotes exhibit genotoxic sensitivities and reduced H2Bub in cis. We therefore conclude that H2BG52D and H2BP102L oncohistones affect HR repair and genome stability via the reduction of H2Bub and propose that other noncanonical oncohistones may also affect histone PTMs to cause diseases.

Conformational switching of Arp5 subunit differentially regulates INO80 chromatin remodeling.

The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80's ATPase activity to nucleosome movement. Our investigation reveals that the grappler domain of Arp5 interacts with the acidic pocket of nucleosomes through two distinct mechanisms: an arginine anchor or a hydrophobic/acidic patch. These two modes of binding serve distinct functions within INO80 as shown in vivo by mutations in these regions resulting in varying phenotypes and in vitro by diverse effects on nucleosome mobilization. Our findings suggest that the hydrophobic/acidic patch of Arp5 is likely important for dimer exchange by INO80, while the arginine anchor is crucial for mobilizing nucleosomes.

Improved detection of DNA replication fork-associated proteins.

Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.

Structure-specific nucleases in genome dynamics and strategies for targeting cancers.

Nucleases are a super family of enzymes that hydrolyze phosphodiester bonds present in genomes. They widely vary in substrates, causing differentiation in cleavage patterns and having a diversified role in maintaining genetic material. Through cellular evolution of prokaryotic to eukaryotic, nucleases become structure-specific in recognizing its own or foreign genomic DNA/RNA configurations as its substrates, including flaps, bubbles, and Holliday junctions. These special structural configurations are commonly found as intermediates in processes like DNA replication, repair, and recombination. The structure-specific nature and diversified functions make them essential to maintaining genome integrity and evolution in normal and cancer cells. In this article, we review their roles in various pathways, including Okazaki fragment maturation during DNA replication, end resection in homology-directed recombination repair of DNA double-strand breaks, DNA excision repair and apoptosis DNA fragmentation in response to exogeneous DNA damage, and HIV life cycle. As the nucleases serve as key points for the DNA dynamics, cellular apoptosis, and cancer cell survival pathways, we discuss the efforts in the field in developing the therapeutic regimens, taking advantage of recently available knowledge of their diversified structures and functions.

Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA.

Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well-studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error-prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C-terminus, as loss of the ImuA C-terminus results in DNA-independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl-imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self-interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the ß-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.

Acute multi-level response to defective de novo chromatin assembly in S-phase.

Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyperaccessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered.

Role of Geminin as a Tool for Augmenting Accurate Diagnosis of Cervical Neoplasia.

AIM: To determine the role of geminin as a tool for differentiating various types of cervical intraepithelial neoplasia (CIN) and cervical carcinoma (CC). METHODS: Seventy women newly diagnosed with CIN or CC undergoing cervical biopsy were included; their clinical profile, human papilloma virus (HPV) positivity, and colposcopy findings were noted, and biopsy tissue was analyzed for geminin content. RESULTS: On geminin immunohistochemistry, 100% of women with CIN3 and 96.29% of women with CC had geminin two plus or more. When analyzed as ordinal variables, there was a significant correlation (spearman's rho 0.35, p 0.01) between geminin and biopsy results (CIN1, CIN2, CIN3, and CC). CONCLUSIONS: Screening tests for cervical cancer, like conventional pap smears, liquid-based pap smears, and triaging with HPV, have limitations. It is important to be able to differentiate between high-grade lesions, invasive cancer, and low-grade lesions. The detection of geminin in these cells may aid in the confirmation of the diagnosis and ensure adequate treatment. Cervical intraepithelial lesions and carcinoma cervix demonstrated a correlation between increased geminin expression in CIN1 vs. CC and CIN2 vs. CC. Geminin may be a potential surrogate marker for higher-grade cervical lesions, and further research is needed to corroborate evidence in this direction.

Maternal methylosome protein 50 is essential for embryonic development in medaka Oryzias latipes.

Methylosome protein 50 (Mep50) is a protein that is rich in WD40 domains, which mediate and regulate a variety of physiological processes in organisms. Previous studies indicated the necessity of Mep50 in embryogenesis in mice Mus musculus and fish. This study aimed to further understand the roles of maternal Mep50 in early embryogenesis using medaka Oryzias latipes as a model. Without maternal Mep50, medaka zygotes developed to the pre-early gastrula stage but died later. The transcriptome of the embryos at the pre-early gastrula stage was analyzed by RNA sequencing. The results indicated that 1572 genes were significantly upregulated and 741 genes were significantly downregulated in the embryos without maternal Mep50. In the differentially expressed genes (DEGs), the DNA-binding proteins, such as histones and members of the small chromosome maintenance complex, were enriched. The major interfered regulatory networks in the embryos losing maternal Mep50 included DNA replication and cell cycle regulation, AP-1 transcription factors such as Jun and Fos, the Wnt pathway, RNA processing, and the extracellular matrix. Quantitative RT-PCR verified 16 DEGs, including prmt5, H2A, cpsf, jun, mcm4, myc, p21, ccne2, cdk6, and col1, among others. It was speculated that the absence of maternal Mep50 could potentially lead to errors in DNA replication and cell cycle arrest, ultimately resulting in cell apoptosis. This eventually resulted in the failure of gastrulation and embryonic death. The results indicate the importance of maternal Mep50 in early embryonic development, particularly in medaka fish.

Ki-67 is necessary during DNA replication for fork protection and genome stability.

BACKGROUND: The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged. Recently, Ki-67 has been shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, how the different roles for Ki-67 across the cell cycle are regulated and coordinated remain poorly understood. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein, coupled to its abundance and fluctuation during the cell cycle. RESULTS: Here, we use a doxycycline-inducible E3 ligase together with an auxin-inducible degron tag to achieve a rapid, acute and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allows us to show that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependent on HUWE1 ligase. This leads to defects in replication and sister chromatids cohesion, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested. CONCLUSIONS: We unveil a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.

Structural Basis for the Interaction of Redß Single-Strand Annealing Protein with Escherichia coli Single-Stranded DNA-Binding Protein.

Redß is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redß is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redß can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redß with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redß and SSB involves the C-terminal domain (CTD) of Redß and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redß CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redß and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redß-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.