APLF and long non-coding RNA NIHCOLE promote stable DNA synapsis in non-homologous end joining
Here, we show that Ku70-Ku80 and APLF establish a minimal complex sufficient to support DNA synapsis. APLF and long non-coding RNA NIHCOLE promote stable DNA synapsis in non-homologous end joining. Additionally, we show how APLF promotes synapsis of DNA ends for several minutes under pN forces and lncRNA NIHCOLE stabilises these synapsis via a small and structured domain relevant in DNA repair by NHEJ.
Continue ReadingHuman HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA
Here, we characterize the biochemical activities of the human DNA helicase B (HELB) using bulk and single-molecule methods.
We found that HELB is a monomeric protein that binds to ssDNA with a site size of 20nt, unexpectedly large for SF1 helicases, suggesting an additional binding site.
HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities.
Translocating HELB concomitantly clears RPA from ssDNA.
A molecular view of DNA flexibility
Here, we review recent single-molecule experiments and molecular dynamics simulations that are providing novel insights into DNA mechanics from such a molecular perspective.
Continue ReadingPurified Smc5/6 Complex Exhibits DNA Substrate Recognition and Compaction
Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities.
Continue ReadingDouble-stranded RNA bending by AU-tract sequences
Sequence-dependent structural deformations of the DNA double helix (dsDNA) have been extensively studied, where adenine tracts (A-tracts) provide a striking example for global bending in the molecule. However, in contrast to dsDNA, sequence-dependent structural features of dsRNA have received little attention. In this work, we demonstrate that the nucleotide sequence can induce a bend in a canonical Watson-Crick base-paired dsRNA helix.
Continue ReadingCharacterizing microfluidic approaches for a fast and efficient reagent exchange in single-molecule studies
Single-molecule experiments usually take place in flow cells. This experimental approach is essential for experiments requiring a liquid environment, but is also useful to allow the exchange of reagents before or during measurements. This is crucial in experiments that need to be triggered by ligands or require a sequential addition of proteins.
Continue ReadingJanuary 2020
January (2020). New paper published in Nucleic Acids Research on the Dynamics of DNA Nicking and Undwinding by the RepC-PcrA complex
January (2020). Alejandro participates on the event “Science in the classroom” by giving a guided tour of the CNB facilities and an introduction of our research work to high school students from the IES ¨Ciudad de los Ángeles¨.
