Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2 Journal Article
| Authors: | Warren, G. M.; Shuman, S. |
| Article Title: | Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2 |
| Abstract: | Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr's ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA. |
| Keywords: | gene deletion; genetics; metabolism; dna damage; dna repair; drug effect; enzymology; bacterial protein; chemistry; bacterial proteins; crystallography, x-ray; mitomycin; x ray crystallography; bacterial dna; dna, bacterial; dna helicase; mycobacterium smegmatis; cross linking reagent; cross-linking reagents; psoralen; dna glycosylase; ap lyase; inter-strand crosslinks; ficusin |
| Journal Title: | mBio |
| Volume: | 15 |
| Issue: | 8 |
| ISSN: | 2150-7511 |
| Publisher: | American Society for Microbiology |
| Date Published: | 2024-08-01 |
| Start Page: | e0124824 |
| Language: | English |
| DOI: | 10.1128/mbio.01248-24 |
| PUBMED: | 39012146 |
| PROVIDER: | scopus |
| PMCID: | PMC11323726 |
| DOI/URL: | |
| Notes: | The MSK Cancer Center Support Grant (P30 CA008748) is acknowledge in the PDF -- Corresponding authors is MSK author: Stewart Shuman -- Source: Scopus |
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