After 30 Years of Study the Bacterial SOS

After 30 Years of Study, the Bacterial SOS Response Still Surprises Us Bénédicte Michel DR Nisreen tashkandy King abdulaziz University

History • Miroslav Radman discovered the DNA repair network in Escherichia coli And introduced the term “SOS response”. • Two proteins : a repressor named Lex. A and an inducer and the Rec. A filament. • During normal growth, the Lex. A repressor binds to the SOS box, present in the promoter region of SOS genes and prevents their expression. • SOS genes are repressed to different degrees under normal growth conditions this depends on the exact sequence of their SOS box , its position in the promoter region, and the strength of the promoter.

'SOS box' • which codes for over 50 genes while Rec. A floats around the cell looking for damaged DNA. • If it finds any, it binds to it and stimulates the breaking of the Lex. A protein. • The SOS box genes are therefore released and the proteins that deal with DNA damage can be made. • Although all the SOS genes are eventually activated, they are not all turned on at once. The first proteins to be made are those that repair simple DNA damage such as problems with single nucleotides.

The SOS response a model for the DNA repair field Treatments of bacterial cultures by a DNA-damaging agent followed by analysis of reporter genes fused to an SOS promoter. Immuno-blottingor for Lex. A or Rec. A proteins. microarrays were used to measure the timing and the amplitude of the induction in bacterial populations. About 40 genes were shown to be under SOS control. Most are DNA repair genes, but there are several genes that still have no known function.

Rec. A • When the cell senses the DNA damage, the Lex. A repressor undergoes a selfcleavage reaction and the SOS genes are activated. • Rec. A is present in nearly all bacteria and conserved in all organisms, including humans the Rec. A filament induces the Lex. A cleavage reaction. • It specifically binds (ss. DNA), forming a nucleoprotein filament that has two functions : the Rec. A filament invade a homologous double-stranded DNA sequence and catalyze strand exchange. • It may promote Lex. A cleavage for inducing the SOS response.

Regulation of Rec. A binding to ss. DNA • the abundant of the ss. DNA binding protein prevent Reca. A from binding to ss. DNA but the Rec. FOR proteins assist Rec. A binding to single-strand gaps. • Rec. BC proteins directly load Rec. A on the processed double-strand ends. • UV light induce the formation of DNA single-strand gaps inducing the SOS response only if : • the Rec. FOR proteins are present • whereas those that create DNA double-strand ends, such as topoisomerase poisons, will require the Rec. BC proteins for SOS induction ends, such as topoisomerase poisons.

DNA topoisomerases play vital roles in DNA replication, transcription, repair and recombination • Topoisomerases removes local topological barriers. Type I topoisomerases cleave and rejoin one strand of DNA. • type II topoisomerases cleave and rejoin a double strand of DNA during catalysis. Bacteria has IIA enzymes are well utilized clinical targets for anticancer and antibacterial chemotherapy. • These topoisomerase targeting compounds initiate the cell killing process by either stabilizing or increasing the accumulation of the covalent complex formed between the enzyme and cleaved DNA and are called topoisomerase poison.

The DNA cleavage reaction of topoisomerase II: wolf in sheep’s clothing • Topoisomerase II is an essential enzyme help in movement of DNA within the nucleus or the opening of the double helix. • This enzyme helps to regulate DNA under and over winding and removes knots and tangles from the genetic material. • In order to carry out its critical physiological functions, topoisomerase II generates transient double-stranded breaks in DNA. • it has the capacity to fragment the genome. The DNA cleavage/ligation reaction of topoisomerase II is the target for some of the most successful anticancer drugs currently in clinical use.

The Rec. BCD enzyme of E. coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA. Protect the bacteria from phages and extraneous chromosomal DNA. regulated by a cis-acting DNA sequence known as Chi that activates its recombination-promoting functions.

Producing a complex sequence-regulated DNA -processing machine. • cis-acting elements are specific binding sites for proteins involved in the initiation and regulation of transcription. • located in the 3′-flanking or downstream region of the transcribed region, or even within the transcribed region, can also influence the initiation of transcription. • Interaction with Chi causes weakening of the Rec. BCD enzyme's strong nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the Rec. A protein onto the resultant Chi-containing single-stranded DNA.

key function of the Rec. BCD enzyme • To rescue broken replication forks via homologous, or template-directed, recombinational DNA repair. • Allows the completion of DNA replication and bacterial cell division. • Degrade unwanted linear duplex DNA that otherwise could block proper replication restart and lead to spurious replication or misplaced recombination.

Excision repair (NER) • After UV irradiation, the amount of Lex. A repressor decreases nearly 10 -fold in a few minutes. • The SOS genes, however, are not all induced at the same time and to the same level. • The first genes to be induced are uvr. A, uvr. B, and uvr. D and endonuclease Uvr. C, catalyze nucleotide excision repair (NER), a reaction that excises the damaged nucleotides from double-stranded DNA.

Homologues recombination • As a second defense against DNA lesions, expression of rec. A and other homologous recombination functions increase more slowly, about 10 -fold. • Homologous recombination allows the repair of lesions that occur on ss. DNA regions at replication forks • The division inhibitor Sfi. A is also induced to give the bacterium time to complete the repairs.

• Finally, about 40 minutes after DNA damage (and if the damage was not fully repaired by NER and homologous recombination), the mutagenic DNA repair polymerase Pol V which encoded from umu. C and umu. D genes is induced. • The last attempt response also allows bacteria to translate DNA lesions double Stranded to be reparable, but at the expense of introducing errors into the genome.

• Clearly, it is important for bacteria to keep all levels of the SOS response under tight control; there is no utility to the organism of using error-prone polymerases longer than absolutely necessary. • lex. A is an SOS gene. The constant production of Lex. A during the SOS process ensures that as soon as DNA repair occurs, the disappearance of the inducing signal will allow Lex. A to re-accumulate and repress the SOS genes. • Moreover, two SOS-induced proteins, Din. I and Rec. X, affect the stability of the Rec. A filament and thus may participate in the control of the SOS response

• in addition to DNA damaging agents, the inactivation of certain cellular functions causes chronic SOS induction. This may be either because the gene product is involved in DNA repair and in its absence spontaneous DNA lesions persist. • Or because inactivated function is essential for proper DNA duplication and the replication defect increases the amount of ss. DNA.

The control of the SOS response • Friedman and coworkers measured in single cells the level and kinetics of activation of SOS promoters after UV-light treatment. • To report promoter activity, the green fluorescent protein (GFP) gene was placed under the control of the promoters of three different SOS genes: • rec. A, lex. A, and umu. CD. • when the signal in a cell population was analyzed, the amount of GFP increased as a broad peak followed by a decrease as repair took place and the SOS response was shut off.

The control of the SOS response; results • in individual cells, one, two, or three consecutive peaks of GFP expression were observed, depending on the UV dose. • At UV doses lower than 10 joules, where most should be removed by NER, one peak of GFP was observed. • This was centered at 20 to 25 minutes after irradiation for the rec. A and the lex. A promoter. • Ten minutes later, the umu. CD promoter was induced. At UV doses of 20 joules or higher, two to three peaks of GFP expression were observed, with the timing of the appearance of the first peak and its amplitude remaining constant.

The control of the SOS response; results • The experiment suggests that in each cell, the SOS response is not simply turned on to an extent that depends on the level of DNA damage and then turned off. • Rather, it suggests that the SOS promoters are induced to a certain level sufficient to survive a certain dose of DNA-damaging agent, regardless of the initial amount of DNA damage. • If the level of DNA damage is too high for the cells to cope with in one round of induction, a second round of induction or even a third round will follow.

The control of the SOS response; results • The interesting finding introduces a whole range of new questions, including: • which factors are limiting the amplitude and controlling the timing of the peaks? • The umu. C and umu. D genes seem to play a role in this process as their inactivation strongly upsets the oscillatory behavior of the rec. A promoter.

SOS and Bacterial Resistance to Antibiotics Beyond being a model of a DNA repair regulatory network, the SOS response has played an important role in shaping the bacterial world. it increases mutations and genetic exchanges. Pol II, Pol IV (din. B), and Pol V (umu. CD) are E. coli SOS-induced DNA polymerases that are able to replicate across lesions (bypass polymerases). only Pol II is induced early and has a high fidelity on intact DNA. In UV-treated cells, these DNA polymerases can induce mutations at the site of the lesion (targeted mutations) or elsewhere (untargeted mutations); their action may be coupled to the repair of DNA double-strand breaks by homologous recombination.

SOS and Bacterial Resistance to Antibiotics The repair of ds DNA breaks necessarily involves a replication, re-initiation step. For example, when wild-type E. coli cells are placed in the presence of a carbon source that they cannot use, some of them suffer double-strand breaks in their chromosomes that are repaired by homologous recombination, creating a substrate for Pol IV. why? Due to the mutagenic action of this DNA polymerase, a sub-population of cells acquires the capacity to use the carbon source and spreads. The mutator effect of Pol III mutations, which affect the main E. coli DNA polymerase and cause chronic SOS induction, also depends in part on the action of SOS-induced polymerases, even in the absence of external damage.

SOS and Bacterial Resistance to Antibiotics the Romesberg laboratory describes the role for SOS induction in the entrance of E. coli mutants resistant to antibiotics. The main antibiotic used is ciprofloxacin, a topoisomerase inhibitor that causes DNA double-strand breaks. The treatment of mice infections by ciprofloxacin leads to the rapid appearance of E. coli cells resistant to the antibiotic. Interestingly, when a pathogenic E. coli strain that encodes a non-cleavable Lex. A repressor is used, no ciprofloxacin-resistant mutant appears.

Inhibition of Mutation and Combating the Evolution of Antibiotic Resistance bacteria evolve resistance to drugs, typically by acquiring chromosomal mutations. bacteria may play a more active role in the mutation of their own genomes in response to at least some DNA-damaging agents by inducing proteins that actually promote mutation. If the acquisition of antibiotic resistance conferring mutations also requires the induction of these proteins, then their inhibition would represent a novel approach to combating the growing problem of drug resistance.

The formation of resistant cells requires SOS induction Recombination and SOS-dependent model is presented for the formation of ciprofloxacin-resistant mutants. Normally, topoisomerase poisoning causes the formation of DNA double-strand breaks, which in turn induce the SOS response and are repaired by homologous recombination. However, when the SOS response is triggered by antibiotic induced DNA damage, the SOS-induced DNA polymerases that act at the replication forks formed by recombination generate mutants some of which are resistant to ciprofloxacin. These findings suggest that blocking SOS induction could be a general means to prevent the rapid evolution of bacteria to antibiotic resistance.

Conclusion Work on the SOS response shows the purpose of bacterial studies, as SOS is both a modulator of bacterial spread during pathogenicity, and an irreplaceable source of concepts for the understanding of DNA repair regulation networks. Several important issues remain to be addressed; 1 - more than a dozen SOS-induced genes encode proteins of unknown function. 2 - identification of their physiological role may reveal new levels or new means of regulation of the SOS response, links with other cellular global regulation networks, and unsuspected consequences of the SOS induction.