Abstract:The bacterial recombinase, RecA protein, plays a central role in maintenance of genome stability. It generally functions as a RecA nucleoprotein filament formed on DNA (1). RecA has three identified functions in the cell. First, it participates directly in all recombination processes. Filaments of Escherichia coli RecA protein form most readily on ssDNA; these filaments can then catalyze DNA pairing and strand exchange with a homologous duplex DNA (2). A RecA filament complexed with an oligonucleotide can inva… Show more
“…In all figures, unless otherwise noted, the variant RecA E38K was used in place of WT RecA. We have previously demonstrated that RecA E38K enhances the RecA-dependent-targeted DSB activity of Ref beyond that of WT RecA (1)
Figure 1.Ref creates targeted DSBs in cdsDNA through a nicked intermediate. ( A ) The reaction scheme is shown.…”
The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein.
“…In all figures, unless otherwise noted, the variant RecA E38K was used in place of WT RecA. We have previously demonstrated that RecA E38K enhances the RecA-dependent-targeted DSB activity of Ref beyond that of WT RecA (1)
Figure 1.Ref creates targeted DSBs in cdsDNA through a nicked intermediate. ( A ) The reaction scheme is shown.…”
The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein.
“…We note that SSPRO8 Figure 2. % coil content vs order counter for sampled molecules, 3PLW 90 (b) 2R6V 91 (c) 1DZF 92 (d) 3HZ8 93 (e) 3UMH 94 . The five proteins are ordered from greatest to least coil content.…”
Section: Resultsmentioning
confidence: 99%
“…We again observe key regions of poor 2D-CNN confidence or inaccuracy and low molecular longevity that do not have a corresponding disorder Figure 6. Models of the five sampled proteins in this study: (i) 3PLW 90 (ii) 2R6V 91 (iii) 1DZF 92 (iv) 3HZ8 93 (v) 3UMH 94 . (a) Molecular longevity on a red/blue scale for low/high structural longevity.…”
Section: Resultsmentioning
confidence: 99%
“…Prediction Accuracy of 5 samples (a) 3PLW90 (b) 2R6V91 (c) 1DZF92 (d) 3HZ893 (e) 3UMH 94 ), with increasing order (sampled proteins).…”
intrinsically disordered proteins (iDps) and intrinsically disordered regions within proteins (iDRs) serve an increasingly expansive list of biological functions, including regulation of transcription and translation, protein phosphorylation, cellular signal transduction, as well as mechanical roles. the strong link between protein function and disorder motivates a deeper fundamental characterization of iDps and iDRs for discovering new functions and relevant mechanisms. We review recent advances in experimental techniques that have improved identification of disordered regions in proteins. Yet, experimentally curated disorder information still does not currently scale to the level of experimentally determined structural information in folded protein databases, and disorder predictors rely on several different binary definitions of disorder. To link secondary structure prediction algorithms developed for folded proteins and protein disorder predictors, we conduct molecular dynamics simulations on representative proteins from the protein Data Bank, comparing secondary structure and disorder predictions with simulation results. We find that structure predictor performance from neural networks can be leveraged for the identification of highly dynamic regions within molecules, linked to disorder. Low accuracy structure predictions suggest a lack of static structure for regions that disorder predictors fail to identify. While disorder databases continue to expand, secondary structure predictors and molecular simulations can improve disorder predictor performance, which aids discovery of novel functions of iDps and iDRs. these observations provide a platform for the development of new, integrated structural databases and fusion of prediction tools toward protein disorder characterization in health and disease.
“…An effort to characterize the bacteriophage P1 Ref protein as a suspected RecA-regulator protein revealed that Ref is actually a RecA-dependent, HNH-family endonuclease ( 7 ). Ref has the novel property of only cleaving DNA to which RecA protein is bound.…”
The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.
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