Inducing DNA-protein crosslinks and 5-azacytidine to study genetic mutation in Escherichia coli

Souradip Mallick, National Institute of Technology, Rourkela

DNA–protein crosslinks induce SOS response as it is mutagenic and 5-azacytidine induces bacterial damage and stimulate homologous recombination. All these consequences of DNA–protein crosslinks and 5-azacytidine were investigated on E.coli

DNA–protein crosslinks (DPCs) are mainly formed when a nucleotide residue on DNA forms a covalent bond with a protein residue. Such type of crosslink is hazardous as it blocks gene transcription and DNA replication. DPCs are the source of DNA damage which is formed by both non-enzymatic and enzymatic mechanisms. DNA topoisomerases and DNA cytosine methyltransferases (CMeTs) are conserved cellular enzymes which are covalently linked to DNA as a reaction intermediate. 

5-Azacytidine, nitrogen analogue of cytidine, exhibits cancerostatic, bacteriostatic as well as some mutagenic properties. It is incorporated into both RNA and DNA but it disrupts protein synthesis. In bacteria, 5-azaC is a useful tool to study the effects of DPCs in vivo whose targets are C5-cytosine methyltransferases (CMeT) in restriction/modification systems. 

The genetic consequences of DPCs formed by cytosine Methyltransferase, Dcm is performed by 5-azacytidine, in E. coli and its following effect was observed. 

DNA damage response: With the help of transcriptional reporter assays for dinBand recA promoters, 5-azacytidine induces the SOS response, which is triggered by the formation of RecA filaments on single-strand DNA in vivo. RecO mutant was reduced, implicating the RecFOR pathway for SOS induction. Thus, in the absence of RecO, Rec BCD pathway also induces SOS.

Homologous recombination:5-azaC induced homologous recombination of about 30-fold which is detected by a gene conversion assay between an internally-deleted lacZ gene and a 500 bp homologous lacZ fragment within the chromosome. The resulting increase in recombination was also seen in a Dcm mutant, proved that lesions other than Dcm-DNA crosslinks can initiate recombination. 

Mutability at QP sites and effects of the SOS response: It is observed that in the absence of RecA, 5-azaC mutagenicity was enhanced at quasi-palindromic (QP) sites, dependent on the Dcm methyltransferase. The elevation by recA was particularly striking for the QP6 reporter for lagging-strand template-switching which is due to lack of the SOS response. The SOS response does have an antimutagenic effect on 5-azaC -induced leading strand QPM.

It is observed that for Dcm-dependent and-independent killing by 5-azaC in sensitive mutants, such as recA, recB, and lon; homologous recombination and deletion mutations are also stimulated in part by a Dcm-independent effect of 5-azaC. But how it occurs that is by a different protein-DNA crosslink or by an alternative form of DNA damage is still unknown. 

Differential effects on template-switching: Although a template-switch mechanism has been observed for both QPM at inverted repeats and deletions/expansion at direct repeats we saw a much stronger effect at inverted repeats. 

Also read: EFFECT OF MISSENSE VARIATIONs: REVELEALED

Source- doi: https://doi.org/10.1101/2020.10.27.357855

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