DNA–protein crosslinks processed by nucleotide excision repair and homologous recombination with base and strand preference in E. coli model system

Abstract

Bis-electrophiles including dibromoethane and epibromohydrin can react with O6-alkylguanine-DNA alkyltransferase (AGT) and form AGT-DNA crosslinks in vitro and in vivo. The presence of human AGT (hAGT) paradoxically increases the mutagenicity and cytotoxicity of bis-electrophiles in cells. Here we establish a bacterial system to study the repair mechanism and cellular responses to DNA-protein crosslinks (DPCs) in vivo. Results show that both nucleotide excision repair (NER) and homologous recombination (HR) pathways can process hAGT-DNA crosslinks with HR playing a dominant role. Mutation spectra show that HR has no strand preference but NER favors processing of the DPCs in the transcribed strand; UvrA, UvrB and Mfd can interfere with small size DPCs but only UvrA can interfere with large size DPCs in the transcribed strand processed by HR. Further, we found that DPCs at TA deoxynucleotide sites are very inefficiently processed by NER and the presence of NER can interfere with these DNA lesions processed by HR. These data indicate that NER and HR can process DPCs cooperatively and competitively and NER processes DPCs with base and strand preference. Therefore, the formation of hAGT-DNA crosslinks can be a plausible and specific system to study the repair mechanism and effects of DPCs precisely in vivo.

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

“…While we have shown that PrimPol re-priming initiates HR at UV, 4-NQO, and BPDE DNA adducts, it will be interesting to investigate whether other bulky lesions or replication impediments can be channeled into this pathway. DNA-protein crosslinks, for example, engage HR for repair 45 , 46 and lead to template-switching 47 raising the question of whether PrimPol could act at these lesions as well. PrimPol also re-primes at R-loops and secondary structure-forming sequences 48 , 49 , opening up the possibility that HR in those backgrounds might depend at least partially on PrimPol.…”

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

“…While we have shown that PrimPol re-priming initiates HR at UV, 4-NQO and BPDE DNA adducts, it will be interesting to investigate whether other bulky lesions or replication impediments can be channelled into this pathway. DNAprotein crosslinks for example engage HR for repair 41,42 and lead to templateswitching 43 raising the question whether PrimPol could act at these lesions as well. PrimPol also re-primes at R-loops and secondary structure-forming sequences 44,45 , opening up the possibility that HR in those backgrounds might depend at least partially on PrimPol.…”

The Cockayne syndrome protein B is involved in the repair of 5-AZA-2′-deoxycytidine-induced DNA lesions

“…Immobilized proteins onto DNA are described to be very effective disruptors of transcription [ 37 ], and are preferentially repaired if they are in the transcribed strand [ 38 ]. The arrest of RNApol II at transcription blocking lesions stops the elongation of the newly synthesized RNA and if the block is prolonged in time, leads into apoptosis.…”