DNA repair and genome maintenance in Bacillus subtilis

Abstract

From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis.

Fig 1

Model for activation of the SOS response in B. subtilis. (A) In this model, UV damage has created a cyclobutane pyrimidine dimer (CPD) in the leading strand template, creating a daughter strand gap after repriming and continued replication beyond the lesion. (B) SSB binds to the daughter strand gap, preserving the integrity of the DNA. (C) Recombinase mediator proteins RecF, RecO, and RecR, and possibly other accessory factors, stimulate RecA loading at the gap region as SSB is displaced from the site. (D) RecA forms a nucleoprotein filament on ssDNA. (E) The RecA-ssDNA nucleoprotein filament then interacts with LexA, activating its latent protease activity and resulting in autocleavage of LexA. Following autocleavage and inactivation of LexA, SOS gene transcription is activated, and a global transcriptional response is induced. (F) SOS-dependent changes in gene expression help B. subtilis to survive DNA damage by upregulating DNA repair proteins, preventing the bacterium from undergoing cell division, and finally increasing the regulatory products RecA and LexA to reset the system after repair is completed. (Adapted from reference .)

Fig 2

Model for repair of a single double-strand break by homologous recombination in B. subtilis. (A) Active replication fork with a single-strand nick in the leading strand template. (B) Upon the fork encountering the lesion, a DSB is produced and the replication fork collapses. (C) The double-stranded end is processed by the AddAB helicase-nuclease complex, or perhaps by RecQ or RecS helicase in combination with RecJ. In this case, AddAB degrades both the 5′ and 3′ ends until it reaches a Chi site, stimulating formation of a 3′-ssDNA tail. (D) The recombinase mediator complex RecFOR is recruited to load the recombinase RecA onto the ssDNA region. This reaction produces a ssDNA-RecA nucleoprotein filament. (E) The RecA-ssDNA filament forms a D loop, where one strand of the template DNA is displaced by the RecA-ssDNA nucleoprotein filament. (F) The 3′ end of the filament is then extended by DNA polymerase by use of the homologous strand as a template for DNA synthesis. The RecG protein or the RuvAB complex can facilitate migration of the D loop. (G) After the damaged strand is sufficiently extended, the Holliday junction is cleaved by RecU or possibly RecV (the dashed line indicates strand nicking). (H) PriA-facilitated replication restart reconstitutes replication of the lagging strand. (Adapted from reference with permission from Elsevier.)

Fig 3

Schematic representation of the domain structure of B. subtilis DNA helicases RecQ and RecS in comparison with human WRN. The N-terminal region contains the helicase motifs (light blue); the RecQ helicase conserved region (RecQ-Ct) (dark blue/purple) and the helicase and RNase D C-terminal domains (HRDC) (red) are also shown. The human protein contains the RNase D domain N-terminal region, which contains a 3′-to-5′ exonuclease domain. (Adapted from references with permission of Oxford University Press, with permission from Elsevier, and with permission from Macmillan Publishers Ltd.)

Fig 4

Model for double Holliday junction formation during homologous recombination and repair of DSBs in B. subtilis. (A) Ionizing radiation or an I-SceI endonuclease creates a DSB in the B. subtilis chromosome. (B) The ends of the DSB are processed by the AddAB helicase-nuclease complex, or perhaps by RecQ or RecS in combination with RecJ. AddAB degrades both the 5′ and 3′ ends until it encounters a Chi site (5′-AGCGG-3′), where 3′-5′ degradation is attenuated, whereas degradation of the 5′-3′ strand continues. This produces a 3′-ssDNA strand on both sides of the DSB, which is bound by SSB. (C) The recombinase mediator complex RecFOR is recruited and functions to load RecA, generating a 3′-ssDNA–RecA nucleoprotein filament. (D) One of the filaments undergoes a homology search and pairs with a template. This produces a displacement loop (D loop) where one strand of the template DNA is displaced by the RecA filament during pairing. One advantage of D loop formation is that the displaced strand can anneal to the other processed DNA, providing a template for its replication. (E) The 3′ ends of both invading strands are then extended by DNA polymerase, using the homologous strand as a template for DNA synthesis. The RecG protein or the RuvAB complex facilitates migration of the D loop, extending the degree of strand exchange. (G) Endonuclease resolution of the double Holliday junctions is facilitated by RecU or RecV, and depending on the location of the cut site, different exchanges between the two strands will be generated. (H) If the Holliday junctions are cleaved at the black dashed line, a gene conversion results in which the flanking sequences are the same as before. (I) If the Holliday junctions are cleaved at the blue dashed line, the downstream sequence flanking the site of damage is exchanged between the two strands. (Adapted from reference with permission from Elsevier.)

Fig 5

Crystal structure of Holliday junction resolvase RecU of B. subtilis. (Adapted from reference with permission from Elsevier.) RecU has been described as a “mushroom”-like protein with a “stalk” and “cap” as indicated in the figure. The catalytic residues critical for Holliday junction cleavage are located in the cap, whereas the stalk interacts with RecA and modulates RecA activity. The Protein Data Bank accession number for the RecU structure is 1ZP7. This image was generated using Pymol (www.pymol.org/).

Fig 6

Model for mismatch repair in B. subtilis. (A and B) The β clamp directs MutS to the DNA to aid in identification of a mismatch. (C) MutS recruits MutL to the site of the mismatch. (D to F) We speculate that the complex slides along the DNA until the latent endonuclease activity of MutL is stimulated, possibly through interaction with the β clamp, causing MutL to nick the nascent strand. (F) The error-containing strand is then unwound, perhaps by RecD2 helicase, and degraded by an exonuclease. New homoduplex DNA is synthesized in the gap, and the new strand is ligated to complete mismatch correction. (Adapted from reference with permission.)

Fig 7

Schematic diagram of the genome maintenance checkpoints in B. subtilis. (A) The interplay between DNA replication and entry into the sporulation program is carefully regulated. The cell cycle regulator protein Sda prevents sporulation when DNA replication is ongoing. In contrast, SirA is activated upon entry into sporulation to prevent new rounds of DNA replication. In addition, entry into sporulation is regulated by at least two separate pathways in response to DNA damage. Sda is upregulated by the SOS response, and DisA senses DNA repair intermediates. Both Sda and DisA inhibit Spo0A phosphorylation, delaying progression of sporulation. (B) B. subtilis has mechanisms that prevent cell division when damaged chromosomes are detected. DnaA regulates cell division by decreasing levels of FtsL, and YneA is an SOS-regulated gene which blocks cell division through an unknown mechanism. These cell cycle checkpoints prevent septum formation and provide cells with a transient period to repair their DNA prior to cytokinesis. Green arrows represent activation events, red line segments represent repression or inactivation, and the black arrow indicates that DNA damage has occurred. (Adapted from reference with permission of the publisher.)