scholarly journals Real-time tracking reveals the catalytic process of Rad51-driven DNA strand exchange

2019 ◽  
Author(s):  
Kentaro Ito ◽  
Yasuto Murayama ◽  
Yumiko Kurokawa ◽  
Shuji Kanamaru ◽  
Yuichi Kokabu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Molecular Model ◽  
Strand Exchange ◽  
Catalytic Process ◽  
Binding Motif ◽  
Binding Motifs ◽  
Dna Binding Motifs ◽  
Dna Strand Exchange ◽  
Motif Site ◽  
Dna Strand

AbstractDuring homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

scholarly journals A serine/proline-rich protein is fused to HRX in t(4;11) acute leukemias

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1124-1131 ◽  
Author(s):  
J Morrissey ◽  
DC Tkachuk ◽  
A Milatovich ◽  
U Francke ◽  
M Link ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Chromosome Band ◽  
Binding Motif ◽  
Significant Similarity ◽  
Acute Leukemias ◽  
Binding Motifs ◽  
Consensus Sequences ◽  
Charged Amino Acids ◽  
Dna Binding Motifs

Translocations involving chromosome band 11q23 in acute leukemias have recently been shown to involve the HRX gene that codes for a protein with significant similarity to Drosophila trithorax. HRX gene alterations are consistently observed in t(4;11) (q21;q23)-carrying leukemias and cell lines by Southern blot analyses and are accompanied by HRX transcripts of anomalous size on Northern blots. HRX-homologous cDNAs were isolated from a library prepared from t(4;11)-carrying acute leukemia cells. cDNAs representative of transcription products from the derivative 11 chromosome were shown to contain HRX sequences fused to sequences derived from chromosome band 4q21. Fragments of the latter were used to clone and analyze cDNAs for wild-type 4q21 transcripts that predicted a 140-Kd basic protein (named FEL) that is rich in prolines, serines, and charged amino acids. FEL contains guanosine triphosphate-binding and nuclear localization consensus sequences and uses one of two possible 5' exons encoding the first 12 or 5 amino acids. After t(4;11) translocations, 913 C-terminal amino acids of FEL are fused in frame to the N-terminal portion of HRX containing its minor groove DNA binding motifs. These features are similar to predicted t(11;19) fusion proteins, suggesting that HRX consistently contributes a novel DNA-binding motif to at least two different chimeric proteins in acute leukemias.

scholarly journals Improved linking of motifs to their TFs using domain information

2019 ◽  
Author(s):  
Nina Baumgarten ◽  
Florian Schmidt ◽  
Marcel H Schulz
Keyword(s):  
Dna Binding ◽  
De Novo ◽  
Domain Score ◽  
Supplementary Information ◽  
P Value ◽  
Binding Motif ◽  
Sequence Information ◽  
Binding Motifs ◽  
Dna Binding Motifs ◽  
Associated Sequences

Abstract Motivation A central aim of molecular biology is to identify mechanisms of transcriptional regulation. Transcription factors (TFs), which are DNA-binding proteins, are highly involved in these processes, thus a crucial information is to know where TFs interact with DNA, and to be aware of the TFs’ DNA-binding motifs. For that reason, computational tools exist that link DNA-binding motifs to TFs either without sequence information or based on TF-associated sequences, e.g. identified via a ChIP-seq experiment. Method In this paper we present MASSIF, a novel method to improve the performance of existing tools that link motifs to TFs relying on TF-associated sequences. MASSIF is based on the idea that a DNA-binding motif, which is correctly linked to a TF, should be assigned to a DNA-binding domain (DBD) similar to that of the mapped TF. Because DNA-binding motifs are in general not linked to DBDs, it is not possible to compare the DBD of a TF and the motif directly. Instead we created a DBD collection, which consist of TFs with a known DBD and an associated motif. This collection enables us to evaluate how likely it is that a linked motif and a TF of interest are associated to the same DBD. We named this similarity measure domain score, and represent it as a p-value. We developed two different ways to improve the performance of existing tools that link motifs to TFs based on TF-associated sequences: (1) using meta analysis to combine p-values from one or several of these tools with the p-value of the domain score and (2) filter unlikely motifs based on the domain score. Results We demonstrate the functionality of MASSIF on several human ChIP-seq data sets, using either motifs from the HOCOMOCO database or de novo identified ones as input motifs. In addition, we show that both variants of our method improve the performance of tools that link motifs to TFs based on TF-associated sequences significantly independent of the considered DBD type. Availability MASSIF is freely available online at https://github.com/SchulzLab/MASSIF Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals R2 Target-Primed Reverse Transcription: Ordered Cleavage and Polymerization Steps by Protein Subunits Asymmetrically Bound to the Target DNA

2005 ◽  
Vol 25 (15) ◽  
pp. 6617-6628 ◽  
Author(s):  
Shawn M. Christensen ◽  
Thomas H. Eickbush
Keyword(s):  
Dna Binding ◽  
Reverse Transcription ◽  
Insertion Site ◽  
Rrna Genes ◽  
28S Rrna ◽  
Protein Subunit ◽  
Binding Motifs ◽  
Dna Binding Motifs ◽  
Target Dna ◽  
Dna Strand

ABSTRACT R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.

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