DarTG is a novel toxin-antitoxin system that catalyses

the reversible ADP-ribosylation of DNA Antonio Ariza1, Gytis Jankevicius1, Marijan Ahel2 and Ivan Ahel1

Summary The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence and programmed cell death. Here we identify and characterise a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis and the thermophilic Thermus aquaticus (Taq). The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADPribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.

ADP-ribosylation is a chemical modification of macromolecules via transfer of an ADP-ribose (ADPr) moiety from NAD+ onto molecular targets (usually proteins). It regulates many processes in eukaryotes and recent studies suggest it may play important roles in bacterial metabolism.

1

We searched for novel ADP-ribosylation systems in bacterial genomes and identified an operon that encodes a conserved protein containing a distinct type of macrodomain (DarG) associated with an uncharacterized protein domain annotated as DUF4433 (DarT).

2

Conclusion

The results of this study have been published as:

The Toxin-Antitoxin System DarTG Catalyzes Reversible ADP-Ribosylation of DNA Jankevicius G *, Ariza A *, Ahel M, Ahel I. Mol Cell. 2016 Dec 15;64(6):1109-1116. doi: 10.1016/j.molcel.2016.11.014.

* Co-first authors Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE Oxford, UK.

E-mail: antonio.ariza@path.ox.ac.uk

ORCID: 0000-0003-4364-823X

Twitter: DrAntonioAriza

Glu + IPTG Ara + IPTG DarG is essential for survival. BL21(DE3) cultures with (A) pBAD TaqDarT E160A and empty pET, (B) pBAD TaqDarT and empty pET, (C) empty pBAD and pET TaqDarG, or (D) pBAD TaqDarG and pET TaqDarG. Expression of pET alone was induced with glucose and IPTG. Expression of both pET and pBAD was induced with arabinose and IPTG.

4 A A

B B C C

D D

DarT and DarG form a complex. SEC elution and SDS-PAGE profiles of TaqDarT E160A, TaqDarG and TaqDarTG complex measured at UV Abs = 280 nm. The elution profiles have been aligned so that fractions from the same elution point correspond vertically with one another and with the SDS-PAGE profiles.

3

Finding potential DarT targets. An activity screen of TaqDarT against various substrates detected by autoradiography of TLC plates after separating the reactions containing 32P-NAD+.

5

DarT ADP-ribosylates ssDNA oligonucleotides. (A) Autoradiography of a denaturing polyacrylamide gel shows TaqDarT modifies short oligonucleotides (GJ1 and GJ1rc) in the presence of 32P-NAD+. (B) UV detection of ethidium bromide-stained denaturing polyacrylamide gels as in (A) in the presence of NAD+.

6

DarG macrodomain de-ADP-ribosylates DarT-ADP-ribosylated oligonucleotides. (A) X-ray co-crystal structures of TaqDarG macrodomain with ADP-ribose. (B) UV detection of ethidium bromide-stained denaturing polyacrylamide gel separating de-ADPribosylation reactions of TaqDarT ADP-ribosylated oligonucleotide by different TaqDarG-macro mutants.

8

DarT ADP-ribosylates a sequence specific thymidine. (A) Mass spectra of 9-mer oligonucleotides. (B) Diagnostic ion magnification of their MS/MS spectra. Relevant fragments on the side and key fragment highlighted in orange. (C) Consensus sequence recognised by DarT.

7 Recognised sequence

DUF4433 Macro18 13015 360209

Toxin AntitoxinScale

1kMycobacterium tuberculosis H37Rvssb rplI Rv0057 dnaB Rv0059 COG2110

Escherichia coli O127:H6 str. E2348/69COG-HsdM COG-HsdS E2348C_1093 COG2110

Pseudomonas mendocina ympCOG-HsdM COG-HsdS Pmen_0565 COG2110

Thermus aquaticus Y51MC23 contigTaqDRAFT_4249 COG2110

Synechocystis sp. PCC 6803 plasmid pSYSGCOG-HsdS sll8002 COG2110

Nitrosomonas europaea ATCC 19718NE1363 COG2110

Klebsiella pneumoniae 342hsdM COG-HsdS KPK_4974 COG2110

darT darG

Proteins

Rv0061 celA1 COG-GlcD

COG610

COG610

COG610

COG610

Substrate

Ø Ø

DN

A

RN

A

Pro

t.

Ø

ADP-ribosylatedproduct

NAD+

TaqDarT WTØ E160AØ

DN

A

RN

A

Pro

t.

DN

A

RN

A

Pro

t.Ø Ø ØWT E160A

Pro

t.

RN

A

DN

A A

DarT - + - + - +GJ1 GJ1rcDNA Ø

ADPr-oligo

B

DarT - + - +GJ1 GJ1rcDNA

ADPr-oligooligo

NAD+

B [a5 - B5] w4[a6 - B6]

m/z1312 1314 1316

%

0

100 1313.31313.3

1314.6m/z

1216 1218 1220

%

0

100 1218.2

1216.2

1219.3

m/z2158 2160

%

0

100 2158.42158.5

2159.52160.7

m/z1216 1218 1220

%

0

100 1218.3

1217.7

1219.2

1219.3m/z

1616 1618 1620

%

0

100 1617.4

1618.4

1619.4

m/z1312 1314 1316

%

0

100 1313.3

1314.31315.3

mod

ified

non-

mod

ified

[M-2H]-2[M-3H]-3

[M-2H]-2[M-3H]-3

m/z900 1000 1100 1200 1300 1400 1500 1600

%

0

100

%

0

100 1066.2

1065.8

929.2

1066.5 1599.81599.3

1066.8

1067.21246.51068.2 1329.2

1600.3

1600.81601.3

1329.3

885.8886.1886.5929.1

1329.8

1330.31330.81340.7

modified M = 3200.42

non-modified M = 2659.46

A

T T T T T C T T T

C

ADPr-GJ1GJ1

Buf

fer

Buf

fer

Buffer7 14 21

WT N22A K29E H82A W83A G119E K80A7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 [min.]

TaqDarG-macro

B

ADPr-oligooligo

DarTG is a novel toxin-antitoxin system andrepresents the first example of reversiblemodification of DNA via ADP-ribosylation. This modification might be more prevalent thanpreviously thought.

A