Sverzhinsky, A. | bioRxiv Science

bioRxiv2024

Inner membrane components of the plasmid pKM101 type IV secretion system TraE and TraD are DNA-binding proteins

The increase of antimicrobial resistance constitutes a significant threat to human health. One of the mechanisms responsible for the spread of resistance to antimicrobials is the transfer of plasmids between bacteria by conjugation. This process is mediated by type IV secretion systems (T4SS) and previous studies have provided in vivo evidence for interactions between DNA and components of the T4SS. Here, we purified TraD and TraE, two inner membrane proteins from the Escherichia coli pKM101 T4SS. Using electrophoretic mobility shift assays and fluorescence polarization we showed that the purified proteins both bind single-stranded and double-stranded DNA in the nanomolar affinity range. The previously identified conjugation inhibitor BAR-072 inhibits TraE DNA binding in vitro, providing evidence for its mechanism of action. Site-directed mutagenesis identified conserved amino acids that are required for conjugation that may be targets for the development of more potent conjugation inhibitors.

biochemistry↗

bioRxiv2021

HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADPribose modifications

Upon detecting DNA strand breaks, PARP1 and PARP2 produce the posttranslational modification poly(ADP-ribose) to orchestrate the cellular response to DNA damage. Histone PARylation factor 1 (HPF1) binds to PARP1/2 to directly regulate their catalytic output. HPF1 is required for the modification of serine residues with ADP-ribose, whereas glutamate/aspartate residues are modified in the absence of HPF1. PARP1 is an abundant nuclear protein, whereas HPF1 is present in much lower amounts, raising the question of whether HPF1 can pervasively modulate PARP1 activity. Here we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at the sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates and dissociates from multiple PARP1 molecules, initiating ADP-ribose modification of serine residues before modification can initiate on glutamate/aspartate residues. HPF1 accelerates the rate of attaching the first ADP-ribose, such that this initiation event is comparable to the rate of the elongation reaction to form poly(ADP-ribose). This "hit and run" mechanism ensures that HPF1 contributions to the PARP1/2 active site during initiation do not persist and interfere with PAR chain elongation at sites of DNA damage. HPF1 thereby balances initiation and elongation events to regulate PARP1/2 output. Structural analysis of HPF1 in complex with PARP1 provides first insights into the assembly on a DNA strand break, and the HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of the serine-ADP-ribose modification in cells and establish that HPF1 imparts the efficiency of serine-ADP-ribose modification required for an acute response to DNA damage.

biochemistry↗