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1. RadD is a RecA-dependent accessory protein that accelerates DNA strand exchange

   https://doi.org/10.1093/nar/gkac041  

   Bonde, Nina J. ; Romero, Zachary J. ; Chitteni-Pattu, Sindhu ; Cox, Michael M. ( February 2022 , Nucleic Acids Research)

   In rapidly growing cells, with recombinational DNA repair required often and a new replication fork passing every 20 min, the pace of RecA-mediated DNA strand exchange is potentially much too slow for bacterial DNA metabolism. The enigmatic RadD protein, a putative SF2 family helicase, exhibits no independent helicase activity on branched DNAs. Instead, RadD greatly accelerates RecA-mediated DNA strand exchange, functioning only when RecA protein is present. The RadD reaction requires the RadD ATPase activity, does not require an interaction with SSB, and may disassemble RecA filaments as it functions. We present RadD as a new class of enzyme, an accessory protein that accelerates DNA strand exchange, possibly with a helicase-like action, in a reaction that is entirely RecA-dependent. RadD is thus a DNA strand exchange (recombination) synergist whose primary function is to coordinate closely with and accelerate the DNA strand exchange reactions promoted by the RecA recombinase. Multiple observations indicate a uniquely close coordination of RadD with RecA function.
2. Interaction with the carboxy-terminal tip of SSB is critical for RecG function in E. coli

   https://doi.org/10.1093/nar/gkad162  

   Bonde, Nina J. ; Henry, Camille ; Wood, Elizabeth A. ; Cox, Michael M. ; Keck, James L. ( March 2023 , Nucleic Acids Research)

   In Escherichia coli, the single-stranded DNA-binding protein (SSB) acts as a genome maintenance organizational hub by interacting with multiple DNA metabolism proteins. Many SSB-interacting proteins (SIPs) form complexes with SSB by docking onto its carboxy-terminal tip (SSB-Ct). An alternative interaction mode in which SIPs bind to PxxP motifs within an intrinsically-disordered linker (IDL) in SSB has been proposed for the RecG DNA helicase and other SIPs. Here, RecG binding to SSB and SSB peptides was measured in vitro and the RecG/SSB interface was identified. The results show that RecG binds directly and specifically to the SSB-Ct, and not the IDL, through an evolutionarily conserved binding site in the RecG helicase domain. Mutations that block RecG binding to SSB sensitize E. coli to DNA damaging agents and induce the SOS DNA-damage response, indicating formation of the RecG/SSB complex is important in vivo. The broader role of the SSB IDL is also investigated. E. coli ssb mutant strains encoding SSB IDL deletion variants lacking all PxxP motifs retain wildtype growth and DNA repair properties, demonstrating that the SSB PxxP motifs are not major contributors to SSB cellular functions.
3. Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD)

   https://doi.org/10.7717/peerj.7724  

   Bayona-Vásquez, Natalia J. ; Glenn, Travis C. ; Kieran, Troy J. ; Pierson, Todd W. ; Hoffberg, Sandra L. ; Scott, Peter A. ; Bentley, Kerin E. ; Finger, John W. ; Louha, Swarnali ; Troendle, Nicholas ; et al ( October 2019 , PeerJ)

   Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed inAdapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility withmore »universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

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4. Detecting genomic deletions from high-throughput sequence data with unsupervised learning

   https://doi.org/10.1186/s12859-023-05139-w  

   Li, Xin ; Wu, Yufeng ( January 2023 , BMC Bioinformatics)

   Structural variation (SV), which ranges from 50 bp to$$\sim$$$\sim$ 3 Mb in size, is an important type of genetic variations. Deletion is a type of SV in which a part of a chromosome or a sequence of DNA is lost during DNA replication. Three types of signals, including discordant read-pairs, reads depth and split reads, are commonly used for SV detection from high-throughput sequence data. Many tools have been developed for detecting SVs by using one or multiple of these signals.

   In this paper, we develop a new method called EigenDel for detecting the germline submicroscopic genomic deletions. EigenDel first takes advantage of discordant read-pairs and clipped reads to get initial deletion candidates, and then it clusters similar candidates by using unsupervised learning methods. After that, EigenDel uses a carefully designed approach for calling true deletions from each cluster. We conduct various experiments to evaluate the performance of EigenDel on low coverage sequence data.

   Our results show that EigenDel outperforms other major methods in terms of improving capability of balancing accuracy and sensitivity as well as reducing bias. EigenDel can be downloaded fromhttps://github.com/lxwgcool/EigenDel.
5. The germline factor DDX4 contributes to the chemoresistance of small cell lung cancer cells

   https://doi.org/10.1038/s42003-023-04444-7  

   Noyes, Christopher ; Kitajima, Shunsuke ; Li, Fengkai ; Suita, Yusuke ; Miriyala, Saradha ; Isaac, Shakson ; Ahsan, Nagib ; Knelson, Erik ; Vajdi, Amir ; Tani, Tetsuo ; et al ( January 2023 , Communications Biology)

   Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DEAD-box helicase 4 (DDX4), a germline factor and RNA helicase conserved in all multicellular organisms, contributes to increased cell motility and cisplatin-mediated drug resistance in small cell lung cancer (SCLC) cells. Proteomic analysis suggests that DDX4 expression upregulates proteins related to DNA repair and immune/inflammatory response. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Further, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of immune/inflammatory response markers. Taken together, we propose that DDX4 increases SCLC cell survival, by increasing the DNA damage and immune response pathways, especially under challenging conditions such as cisplatin treatment.