Dimitra Markovitsi replied: Your papers have indeed been very stimulating for our studies. However, in our experiments, using much lower excitation intensities (<4 mW cm À2) than yours (>4 mW cm À2), we found that the one-photon ionization of mono-nucleosides and mono-nucleotides is lower than 3 Â 10 À4 , which corresponds to our detection limit. In contrast, when using similar excitation intensities to those reported in your 2002 paper we do observe ionization from the buffer. The fact that we observe one-photon ionization only for oligomers Faraday Discussions Discussions

Amitabha Chattopadhyay opened the discussion of the paper by Gebhard F. X. Schertler: Do you have an idea of the timescale of the dynamics of these water molecules in GPCR structures? Gebhard F. X. Schertler replied: This depends on the water molecules that you are referring to. The water molecules close to the retinal will respond very fast. From looking at the time resolved measurements with bacteriorhodopsin, we know that it is faster than 16 nanoseconds. I expect it to be on the picosecond timescale. Water molecules in the hydrogen bond network connecting to the G protein binding site will rearrange in the same time frame as the conformational changes. In rhodopsin this ranges from microseconds to milliseconds. Richard Cogdell remarked: I agree that water molecules are important. Why do you think you would use water in this space rather than a larger amino acid? Water gives you a much greater structural exibility and a faster timescale. Do you think that is a reasonable way to think about it? Gebhard F. X. Schertler responded: As I pointed out, water molecules can have many roles inside a protein. I focused on the highly occupied water sites. This gives little information about the dynamics and exchange rates of the water molecules. It also does not mean that less ordered water molecules inside the protein are not important and so they could exist if the space allows it. I think you are implying

No abstract

Single-molecule studies of protein-nucleic acid interactions frequently require sitespecific modification of long DNA substrates. DNA isolated from bacteriophage λ (λ-DNA) is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific DNA sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multistep ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into λ-DNA with 90-100% yield. Furthermore, various DNA structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of Proliferating Cell Nuclear Antigen (PCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that PCNA can load on both 5'-ssDNA flaps and a 13xCAG triplet repeat. However, PCNA remains trapped on the 13xCAG structure, confirming a proposed mechanism for triplet repeat expansion. Although we focus on λ-DNA, this method is applicable to all long DNA substrates. We anticipate that this molecular toolbox will be broadly useful for both ensemble-and singlemolecule studies that require site-specific modification of long DNA substrates.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.