The recombination initiation functions DprA and RecFOR suppress microindel mutations in Acinetobacter baylyiADP1

Abstract

Short‐Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single‐strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single‐strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single‐strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR‐avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non‐transformable bacteria.

Plasmids modulate microindel mutations inAcinetobacter baylyiADP1

“…We repeated the experiments in the recJ + exoX + ("wildtype") A. baylyi hisC::'ND5i' strain. In this strain, SPDIR frequencies are generally about 30-fold lower than in a ΔrecJ ΔexoX background (Harms et al, 2016;Liljegren et al, 2024), precluding significance statements due to zero inflation, but the tendencies were very similar to the results observed in the ΔrecJ ΔexoX strain. Without plasmid, the median His + frequency was the lowest, and no SPDIR mutant was recovered.…”

“…The mutation assays were conducted as reported previously (Liljegren et al, 2024) and illustrated in Fig. 3B.…”

“…It was recently shown that ssDNA can be mutagenic through the Short-Patch Double Illegitimate Recombination (SPDIR) pathway (Harms et al, 2016; Liljegren et al, 2024). SPDIR mutations are caused by ssDNA from intragenomic or external sources (e.g., acquired in the course of horizontal gene transfer), and those ssDNA molecules can anneal with transiently single-stranded chromosomal DNA sections, such as lagging strands at replication forks.…”

“…It is thought that these annealed ssDNA molecules are extended and eventually integrated into the nascent strand during genomic DNA replication (Fig. 1A), acting in effect as primers for Okazaki fragments (Harms et al, 2016; Liljegren et al, 2024). The resulting mutations are nucleotide polymorphism clusters or microindels of highly variable size and sequence, and the mutation patterns can be traced back to the DNA sequence of the templating single-strand (Fig.…”

“…SPDIR mutations have been found to be very rare (between 10 -12 and 10 -13 per locus and cell in the gamma-proteobacterial model organism Acinetobacter baylyi ; Liljegren et al, 2024). The SPDIR mutation mechanism is probably widespread and potentially universal, and SPDIR mutations have been identified retrospectively in silico in the Gram-positive Streptococcus pneumoniae and in the human genome as well (Harms et al, 2016).…”