RecA finds homologous DNA by reduced dimensionality search

Abstract

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3′ single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.

Different modes of spacer acquisition by the Staphylococcus epidermidis type III-A CRISPR-Cas system

“…However, using our inducible spacer acquisition assay, we were able to demonstrate that the spacers acquired from this region by both the S. epidermidis type III-A and the S. pyogenes type II-A CRISPR systems, mediate inefficient self-targeting, as cells harboring these spacers are not negatively selected after prolonged induction of spacer acquisition. A possible explanation for the lack of targeting could be the abundance of DNA repair machinery as well as ssDNA templates for homologous recombination at the dif site ( 51 ), which could efficiently fix the DNA damage inflicted by the crRNA-guided Cas nuclease. If so, this would represent an evolved mechanism of the host to lower the cost of CRISPR autoimmunity at the chromosomal terminus.…”

Speciation of Genes and Genomes: Conservation of DNA Polymorphism by Barriers to Recombination Raised by Mismatch Repair System

“…See text and refs. (Shen and Huang, 1986;Radman, 1989;Kowalczykowski et al, 1994;Worth et al, 1994;Štambuk and Radman, 1998;Wiktor et al, 2021 apparently irreversible, whereas recombination between nonidentical sequences is apparently mostly reversible (Worth et al, 1994). This is the general principle of the kinetic proofreading theory (off/on rate ratio is increased for wrong substrates and intermediates in biosynthetic processes).…”

“…To initiate homologous recombination, the recombination machinery (protein complex associated with the prototypic homolog of the bacterial RecA recombinase) requires a minimum length of strict sequence identity, called MEPS (for minimal efficient processing segment) ( Shen and Huang, 1986 ). MEPS corresponds to the length of single-stranded DNA that—as RecA nucleofilament—searches the nucleus (or bacterial nucleoid) for duplex DNA stretch of identical sequence to initiate strand exchange ( Wiktor et al, 2021 ). Bacterial MEPS is a matching sequence of, at most, 30 identical base pairs ( Shen and Huang, 1986 ), whereas eukaryotic MEPS length is in the range between 200 (in lower eukaryotes) and 400 (in mammals) nucleotides ( Kricker et al, 1992 ; Kowalczykowski et al, 1994 ; Štambuk and Radman, 1998 ).…”

“…See text and refs. ( Shen and Huang, 1986 ; Radman, 1989 ; Kowalczykowski et al, 1994 ; Worth et al, 1994 ; Štambuk and Radman, 1998 ; Wiktor et al, 2021 ).…”

Searching for DNA Damage: Insights From Single Molecule Analysis

“…Instead of one straight piece of DNA (i.e., as a tightrope), damage detection proteins must sift through many loops and coils of genomic DNA, often bound by various other factors (Figure 4). Hence, a growing body of studies has been aimed at understanding how proteins function at the single molecule level within living cells, including recent work showing double-strand DNA break repair in living E. coli (Lawaree et al, 2020;Wiktor et al, 2021). Efforts at observing NER at the single molecule scale within living cells have begun with multiple studies imaging prokaryotic NER damage detecting proteins UvrA and UvrB in E. coli (Stracy et al, 2016;Springall et al, 2018;Ghodke et al, 2020;Ho et al, 2020).…”