Abstract
Site-specific recombination is a DNA breaking and reconstructing process that plays important roles in various cellular pathways for both prokaryotes and eukaryotes. This process requires a site-specific recombinase and direct or inverted repeats. Some tyrosine site-specific recombinases catalyze DNA inversions and regulate subpopulation diversity and phase variation in many bacterial species. In Streptococcus pneumoniae, the PsrA tyrosine recombinase was shown to control DNA inversions in the three DNA methyltransferase hsdS genes of the type I restriction-modification cod locus. Such DNA inversions are mediated by three inverted repeats (IR1, IR2, and IR3). In this work, we purified an untagged form of the PsrA protein and studied its DNA-binding and catalytic features. Gel retardation assays showed that PsrA binds to linear and supercoiled DNAs, containing or not inverted repeats. Nevertheless, DNase I footprinting assays showed that, on linear DNAs, PsrA has a preference for sites that include an IR1 sequence (IR1.1 or IR1.2) and its boundary sequences. Furthermore, on supercoiled DNAs, PsrA was able to generate DNA inversions between specific inverted repeats (IR1, IR2, and IR3), which supports its ability to locate specific target sites. Unlike other site-specific recombinases, PsrA showed reliance on magnesium ions for efficient catalysis of IR1-mediated DNA inversions. We discuss that PsrA might find its specific binding sites on the bacterial genome by a mechanism that involves transitory non-specific interactions between protein and DNA.
Highlights
Site-specific recombination (SSR) is a DNA breaking and reconstructing process widely distributed in both prokaryotes and eukaryotes, in which a specialized enzyme catalyzes reciprocal strand exchange at specific target sites
The gene encoding the PsrA Tyr-recombinase is included within the cod locus. in vivo studies showed that PsrA plays an essential role in promoting hsdS inversions between a pair of IR1 or IR1-like inverted repeats, with the exception of low-frequency sequenceand RecA-independent “spontaneous” inversions (Li et al, 2019)
In DNA-binding proteins, the location of their target sites on the genome is often preceded by an extensive search process, which results in transitory non-specific interactions between protein and DNA (Halford and Marko, 2004; Marcovitz and Levy, 2013)
Summary
Site-specific recombination (SSR) is a DNA breaking and reconstructing process widely distributed in both prokaryotes and eukaryotes, in which a specialized enzyme catalyzes reciprocal strand exchange at specific target sites. According to this fundamental definition, two critical elements participate in the process, namely a site-specific recombinase and a pair of inversely or directly repeated sequences (Grindley et al, 2006; Rajeev et al, 2009). In addition to the difference in the catalytic residues, the catalytic mechanisms of Tyr- and Ser-recombinases are different In the former, after the nucleophilic attack, the catalytic Tyr residue remains covalently linked to the 3′-end of the DNA strand, generating a 3′-phospho-tyrosyl bond and leaving a free 5′-OH group. Unlike general homologous recombination event that requires large-size homologous DNA segments, DNA replication and high-energy cofactors, the specific repeated sequences recognized by site-specific recombinases are usually 20–40 bp in length, and no DNA synthesis or energy factors are required (Grindley et al, 2006)
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