Biology subjects

Kaplan, A.

Publications and source records attributed to Kaplan, A..

5 recordsLinked to original sources

bioRxiv2017

Auxiliary ATP binding sites power rapid unwinding by RecBCD

RecBCD, responsible for the initiation of double stranded break repair in bacteria, is a processive DNA helicase with an unwinding rate approaching [~]1,600 bp{middle dot}s-1. The mechanism enabling RecBCD to achieve such fast unwinding rate is not known. We employed a combination of equilibrium and time-resolved binding experiments, and ensemble and single molecule activity assays to uncover the molecular mechanism underlying RecBCDs rapid catalysis. We report the existence of auxiliary binding sites, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. The catalytic rate of RecBCD is reduced both by preventing and by strengthening ATP binding to these sites, suggesting that the dynamics of ATP at these sites modulates the enzymes rate. We propose a model by which RecBCD achieves its fast unwinding rate by utilizing the weaker binding sites to increase the flux of ATP to its catalytic sites.

biophysics↗

bioRxiv2017

The mechano-chemistry of reverse transcriptase

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.

biophysics↗

bioRxiv2017

RecBCD possesses strong coupling between DNA and nucleotides binding that may propel stepping mechanism during translocation

Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping essential for break repair. Yet, the energetics of RecBCD during translocation along the DNA track needs to be quantitatively clarified. Specifically, its essential to understand how RecBCD switches between its binding states to enable its translocation. Here we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. We show that, in the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to "sense" the nucleotide state. Excitingly, we observed weak coupling for AMPpNp as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of RecBCD reaction mechanism suggests an energetic compensation between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a stepping mechanism during the processive translocation of RecBCD.

biochemistry↗

bioRxiv2017

Single-molecule DNA unzipping reveals asymmetric modulation of the transcription factor Egr-1 by its binding site sequence and context

Most functional transcription factor (TF) binding sites deviate from their \"consensus\" recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 base pairs bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

biophysics↗

bioRxiv2017

Mobile nucleosomes and the accessibility of transcription factors to DNA: a modified dynamic equilibrium model

Nucleosomes, the basic building block of chromatin, regulate the accessibility of the transcription machinery to DNA. Recent studies have revealed that the nucleosome's spontaneous, thermally driven positional dynamics are modulated by different factors, and exploited by the cell as a regulatory mechanism. In particular, enrichment of mobile nucleosomes at the promoters of genes suggests that the mobility of nucleosomes may affect the ability of transcription factors to bind DNA. However, a quantitative model describing the effect nucleosome mobility on the effective affinity of transcription factors is lacking. We present here a simple equilibrium model that captures the essence of the effect, and show that modulation of the nucleosome's mobility can be a potent and versatile regulator of transcription factor binding.

biophysics↗